Combination therapies comprising anti-erbb3 agents

ABSTRACT

Disclosed are methods and compositions for inhibiting the growth of a tumor (e.g., a malignant 5 tumor) in a subject. In particular, combination therapies for treating a tumor in a subject by coadministering an agent selected from i) an effective amount of an anti-estrogen agent; ii) an effective amount of a receptor tyrosine kinase inhibitor; iii) an effective amount of a MEK/PI3 kinase/AKT inhibitor; iv) an effective amount of MM-151; v) an effective amount of an mTOR inhibitor; and/or vi) an effective amount of trastuzumab or T-DM 1, and/or combinations thereof; and an effective amount of a 10 bispecific anti-ErbB2/anti-ErbB3 antibody.

FIELD OF THE INVENTION

The various aspects of the invention disclosed herein relate to methodsand compositions for the treatment of cancers.

BACKGROUND OF THE INVENTION

Approximately 75% of breast cancers are estrogen receptor (ER) positive.Other cancers are also ER positive (ER+). Estrogen receptors mediateintracellular signaling that can increase the frequency of cell divisionand drive tumor growth. Although anti-endocrine therapies such astamoxifen, fulvestrant, and letrozole have demonstrated significantefficacy in treating ER+ breast cancer patients, intrinsic or acquiredresistance to such therapies has limited their success.

The prevalence of amplification of the human epidermal growth factorreceptor 2 (HER2, or ErbB2) in breast cancer and other cancers hasresulted in the research and development of drugs that have ErbB2 as atherapeutic target. Although both the anti-ErbB2 monoclonal antibodytrastuzumab, the anti-ErbB2 monoclonal antibody drug conjugate T-DM1(ado-trastuzumab emtansine) and the ErbB1/ErbB2 dual receptor tyrosinekinase inhibitor lapatinib have met with success in the clinic, manypatients fail to benefit from these drugs. Additionally, the majority ofpatients with tumors that initially respond will eventually recrudesceafter extended treatment using these therapies.

ErbB2 can function as a homodimer or as a heterodimer with relatedepidermal growth factor-type receptors such as ErbB3. The ErbB2/ErbB3heterodimer is the most potent ErbB receptor pairing with respect tostrength of interaction, impact on receptor tyrosine phosphorylation,and effects on downstream intracellular signaling through mitogenactivated protein kinase and phosphoinositide-3 kinase pathways.Heregulin is the primary ligand for ErbB3, and activates signaling byErbB2/ErbB3 heterodimers. Such signaling is believed to driveproliferation of cancer cells. Current ErbB2-targeted therapies do noteffectively inhibit heregulin activated signaling. MM-111 is abispecific anti-ErbB2/anti-ErbB3 antibody that abrogates heregulinbinding to ErbB2/ErbB3 and inhibits heregulin activation of ErbB2/ErbB3without significantly affecting ErbB2 biological activity. Inpreclinical models of HER-2+ gastric, breast, ovarian and lung cancers,MM-111 inhibits ErbB3 phosphorylation, cell cycle progression, and tumorgrowth.

Thus, a need exists for therapies and therapeutic strategies providingimproved inhibition of ErbB3 activation (e.g., ligand-inducedactivation) as well as for therapies and therapeutic strategiesproviding improved inhibition of estrogen receptor signaling activity orof ErB1 and ErbB2 receptor signaling activity, as well as inhibition ofthe downstream intracellular signaling pathways such as the mitogenactivated protein kinase and phosphoinositide-3 kinase pathways.

In the treatment of cancers, the co-administration of pluralities ofanti-cancer drugs (combination therapy) often provides better treatmentoutcomes than monotherapy. Such outcomes can be subadditive, additive,or superadditive. That is to say that the combined effects of twoanti-cancer drugs, each of which provides a quantifiable degree ofbenefit, can be less than, equal to, or greater than the sum of thebenefits of each drug. For example, two drug, each of which when usedalone to treat a lethal cancer provides an average one year extension ofprogression free survival, could together provide a <24 month extension(e.g., an 18 month extension), about a 24 month extension, or a >24month extension (e.g., a 30 month extension) of progression freesurvival. Typically, combination therapies for cancer treatment providesignificantly subadditive outcomes. Outcomes that are near additive,additive, or superadditive are most desirable, but only occur rarely. Inaddition, many drugs are known to alter the bioavailability, orotherwise affect the safety profile of other drugs when both drugs areco-administered. As new drugs are first used in combination therapies,unforeseen, hazardous drug-drug interactions may be observed that resultin drug-drug interaction-mediated toxicity in the patient.

Thus approaches for safely administering combination therapiescomprising administration of ErbB2/ErbB3 heterodimer-targeted agents forcancer treatment, and especially combinations that yield near-additive,additive, or superadditive outcomes are needed.

SUMMARY OF THE INVENTION

Provided herein are methods and compositions effective for theinhibition of ErbB3 activation and also effective for the inhibition ofestrogen receptor activation. Also provided are methods and compositionseffective for the inhibition of ErbB3 activation and also effective forthe inhibition of ErB1 and/or ErbB2 activation. These methods andcompositions are useful for the treatment of tumors, e.g., malignanttumors, as well as for the treatment of other cancers.

In a first embodiment, a method of treating a subject with a malignanttumor is provided, where the tumor is an ErbB2 expressing or ErbB2over-expressing tumor (e.g., HER2⁺⁺ or HER2⁺⁺⁺ tumors) and the tumor maybe a melanoma, clear cell sarcoma, head and neck, endometrial, prostate,breast, ovarian, esophageal, gastric, gastro-esophageal, colon,colorectal, lung, bladder, pancreatic, salivary gland, liver, skin,brain or renal tumor. The method comprises co-administering to thesubject an effective amount an agent selected from i) an anti-estrogenagent, ii) a receptor tyrosine kinase inhibitor, iii) a MEK inhibitor,(e.g., selumetinib, trametinib, PD0325901, UO126), iv) a PI3 kinaseinhibitor (e.g., BEZ235, BKM120, GDC0941), v) an AKT inhibitor (e.g.,MK-2206, triciribine), vi) an mTOR inhibitor (e.g., BEZ235, AZD8055,everolimus, temsirolimus, sirolimus, ridaforolimus), vii) trastuzumab,viii) T-DM1, ix) capecitabine, x) cisplatin, and xi) MM-151; andcombinations thereof, in combination with an effective amount of ananti-ErbB3 agent, e.g., a bispecific anti-ErbB2/anti-ErbB3 antibody(e.g., an antibody comprising the amino acid sequence set forth in SEQID NO:1). Additional agents for use in combination with the anti-ErbB3agent, e.g., a bispecific anti-ErbB2/anti-ErbB3 antibody, are describedin the Appendix.

In one aspect, the combination of the bispecific anti-ErbB2/anti-ErbB3antibody and either the effective amount of an anti-estrogen agent orthe effective amount of the receptor tyrosine kinase inhibitor, andoptionally the effective amount of trastuzumab or T-DM1, ischaracterized as follows: when a first tissue culture medium is preparedcomprising the bispecific anti-ErbB2/anti-ErbB3 antibody (e.g., theantibody comprising the amino acid sequence set forth in SEQ ID NO:1) ata first concentration and either the anti-estrogen agent at a secondconcentration or the receptor tyrosine kinase inhibitor (e.g.,lapatinib) at a third concentration (wherein each concentration is thesame or different as each other concentration), and the medium iscontacted with cancer cells of a cell line in a cell culture, cellgrowth or cell proliferation or production of pErbB3 or production ofpAKT in the cells is inhibited, or the percentage of cells in theculture that are apoptotic is increased. In certain aspects, cell growthor cell proliferation or production of pErbB3 or production of pAKT inthe cells is inhibited, or the percentage of cells in the culture thatare apoptotic is increased to a greater degree than cell growth, or cellproliferation or production of pErbB3 or production of pAKT in the cellsis inhibited, or percentage of cells in the culture that are apoptoticis increased, to a lesser degree when cancer cells of the cell line in acell culture are contacted with each of a second medium that isessentially the same as the first medium except that it does notcomprise a bispecific anti-ErbB2/anti-ErbB3 antibody, and a third mediumthat is essentially the same as the first medium except that it does notcomprise any anti-estrogen agent and it does not comprise any receptortyrosine kinase inhibitor.

In another aspect, all effective amounts are either mouse effectiveamounts or human effective amounts. In another aspect, all effectiveamounts are mouse effective amounts and the combination of thebispecific anti-ErbB2/anti-ErbB3 antibody (optionally the antibodycomprising the amino acid sequence set forth in SEQ ID NO:1) and eitherthe effective amount of an anti-estrogen agent or the effective amountof the receptor tyrosine kinase inhibitor, is characterized as follows:when co-administered to BT474-M3 xenograft tumor bearing mice with atumor of a measured volume, the combination is more effective atinhibiting tumor volume increase after 32 days of co-administration thanis the mouse effective amount of the bispecific anti-ErbB2/anti-ErbB3antibody administration without the co-administration of either theeffective amount of an anti-estrogen agent or the effective amount ofthe receptor tyrosine kinase inhibitor. In another aspect, a mouseeffective amount of trastuzumab or T-DM1 is co-administered with thebispecific anti-ErbB2/anti-ErbB3 antibody.

In a second embodiment, a bispecific anti-ErbB2/anti-ErbB3 antibody(optionally the antibody comprising SEQ ID NO:1) is provided for use incombination therapy of a cancer (optionally a melanoma, clear cellsarcoma, head and neck, endometrial, esophageal, gastro-esophagealjunction, prostate, breast, ovarian, gastric, colon, colorectal, lung,bladder, pancreatic, salivary gland, liver, skin, brain or renal tumor),where the combination therapy comprises concomitant use of an effectiveamount an agent selected from i) an anti-estrogen agent, ii) a receptortyrosine kinase inhibitor, iii) a MEK inhibitor, iv) a PI3 kinaseinhibitor, v) an AKT inhibitor, vi) an mTOR inhibitor, vii) trastuzumab,viii) T-DM1, ix) capecitabine, x) cisplatin, and xi) MM-151; andcombinations thereof.

In a third embodiment, an aqueous solution is provided comprising abispecific anti-ErbB2/anti-ErbB3 antibody (optionally an antibodycomprising the amino acid sequence set forth in SEQ ID NO:1) at a firstconcentration and an agent selected from i) an anti-estrogen agent, ii)a receptor tyrosine kinase inhibitor, iii) an effective amount of a MEKinhibitor, iv) a PI3 kinase inhibitor, v) an AKT inhibitor, vi) an mTORinhibitor, vii) trastuzumab, viii) T-DM1, ix) capecitabine, x)cisplatin, and xi) MM-151; and combinations thereof, at a secondconcentration. In certain aspects, when a first tissue culture medium isprepared comprising the bispecific anti-ErbB2/anti-ErbB3 antibody at thefirst concentration and the agent at the second concentration, and themedium is contacted with cancer cells of a cell line in a cell culture,cell growth or cell proliferation or production of pErbB3 or productionof pAKT in the cells is inhibited, or percentage of cells in the culturethat are apoptotic is increased. In certain aspects, cell growth or cellproliferation or production of pErbB3 or production of pAKT in the cellsis inhibited, or the percentage of cells in the culture that areapoptotic is increased to a lesser degree when cells of the cell line ina cell culture are contacted with a second tissue culture medium that isessentially the same as the first medium of except that it does notcomprise the agent(s). In another aspect, cell growth or cellproliferation or production of pErbB3 or production of pAKT in the cellsis inhibited, or the percentage of cells in the culture that areapoptotic is increased to a lesser degree when cells of the cell line ina cell culture are contacted with a third tissue culture medium that isessentially the same as the first medium of except that it does notcomprise any bispecific anti-ErbB2/anti-ErbB3 antibody.

In another aspect, the aqueous solution is blood plasma in a subject,and the subject does not experience a toxicity that is sufficientlyharmful to require a change in a therapy being administered to thesubject, which toxicity is mediated by a drug-drug interaction in thesubject between the bispecific anti-ErbB2/anti-ErbB3 antibody and theanti-estrogen agent or the receptor tyrosine kinase inhibitor.

In another aspect, the aqueous solution further comprises trastuzumab orT-DM1 at a third concentration.

In another aspect, the method, combination therapy, or aqueous solutiondoes not comprise an aromatase inhibitor or an estrogen receptorantagonist. In one embodiment the method, combination therapy, oraqueous solution comprises nab-paclitaxel.

In each embodiment and aspect thereof above, the anti-estrogen agent maybe an estrogen receptor antagonist (e.g., fulvestrant or tamoxifen) oran aromatase inhibitor (e.g., wherein the aromatase inhibitor isletrozole, exemestane, anastrozole, aminoglutethimide, testolactone,vorozole, formestane, or fadrozole. Preferably the aromatase inhibitoris letrozole. Also in each embodiment and aspect thereof above, thereceptor tyrosine kinase inhibitor is erlotinib, afatinib, dasatinib,gefitinib, imatinib, pazopinib, lapatinib, sunitinib, nilotinib orsorafenib. Preferably the receptor tyrosine kinase inhibitor islapatinib. Also in each embodiment and aspect thereof above, thebispecific anti ErbB2/anti-ErbB3 antibody is the A5-HSA-ML3.9,ML3.9-HSA-A5, A5-HSA-B1D2, B1D2-HSA-A5, B12-HSA-B1D2, B1D2-HSA-B12,A5-HSA-F5B6H2, F5B6H2-HSA-A5, H3-HSA-F5B6H2, F5B6H2-HSA-H3,F4-HSA-F5B6H2, F5B6H2-HSA-F4, B1D2-HSA-H3, H3-HSA-B1D2, or the antibodycomprising the amino acid sequence set forth in SEQ ID NO:1. Eachembodiment and aspect thereof above may also further comprise use ofcapecitabine and/or cisplatin.

In each embodiment and aspect thereof above, one or more of a)-i) thatfollow may optionally apply: a) the cell line is BT474-M3; b) theculture is a spheroid culture, c) paclitaxel or another taxane oranother chemotherapeutic drug is co-administered, optionally inaccordance with the manufacturer's directions, d) the agent i)-xi) isadministered in accordance with the manufacturer's directions, e) thetrastuzumab or T-DM1 is administered in accordance with themanufacturer's directions, f) the co-administration of the bispecificanti-ErbB2/anti-ErbB3 antibody with the agent g)-vi) produces an aboutadditive or a superadditive effect, h) the bispecificanti-ErbB2/anti-ErbB3 antibody is the antibody comprising SEQ ID NO:1and is administered in accordance with any of the regimens (e.g., modes,dosages, dosing intervals, loading and maintenance doses and dosingschemes) described in Examples 12 and 13, below, i) the lapatinib isadministered in accordance with any of the regimens (e.g., modes,dosages, dosing intervals, loading and maintenance doses and dosingschemes) described in Example 16, below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing that the combination of MM-111 and tamoxifeninhibits tumor growth in vivo better than either MM-111 or tamoxifendoes alone. The x-axis shows time post tumor implant in days and they-axis shows tumor volume in mm³. Mice were treated with inhibitorsbeginning on day 7 post BT474-M3 cell implant.

FIGS. 2A-2G are seven graphs showing that MM-111 combines positivelywith anti-estrogen drugs in inhibiting estrogen-stimulated spheroidgrowth in vitro. FIG. 2A shows the effect of MM-111, tamoxifen(4-hydroxytamoxifen or 4OHT), or MM-111 and tamoxifen on in vitrospheroid growth.

FIG. 2B shows the effect of trastuzumab, tamoxifen, or trastuzumab andtamoxifen. FIG. 2C shows the effect of MM-111, fulvestrant (FVT), orMM-111 and fulvestrant. FIG. 2D shows the effect of trastuzumab,fulvestrant, or trastuzumab and fulvestrant. FIG. 2E shows the effect ofMM-111, trastuzumab, or MM-111 and trastuzumab. FIG. 2F shows the effectof MM-111, trastuzumab, and tamoxifen combined compared to that of anyof the double combinations. FIG. 2G shows the effect of MM-111,trastuzumab, and fulvestrant combined compared to that of any of thedouble combinations. The x-axes are a log scale of each drugconcentration for each experimental condition in nM and the y axis isspheroid size as % of control spheroid size.

FIGS. 3A-3G are seven graphs showing that MM-111 combines positivelywith anti-estrogen drugs in inhibiting heregulin (HRG)-stimulatedspheroid growth in vitro. FIG. 3A shows the effect of MM-111, tamoxifen(4-hydroxytamoxifen or 4OHT), or MM-111 and tamoxifen. FIG. 3B shows theeffect of trastuzumab, tamoxifen, or trastuzumab and tamoxifen. FIG. 3Cshows the effect of MM-111, fulvestrant (FVT), or MM-111 andfulvestrant. FIG. 3D shows the effect of trastuzumab, fulvestrant, ortrastuzumab and fulvestrant. FIG. 3E shows the effect of MM-111,trastuzumab, or MM-111 and trastuzumab. FIG. 3F shows the effect ofMM-111, trastuzumab, and tamoxifen combined compared to that of any ofthe double combinations. FIG. 3G shows the effect of MM-111,trastuzumab, and fulvestrant combined compared to that of any of thedouble combinations. The x-axes are a log scale of each drugconcentration for each experimental condition in nM and the y axis isspheroid size as % of control spheroid size.

FIGS. 4A-4G are seven graphs showing that MM-111 combines positivelywith anti-estrogen drugs in inhibiting dual ligand (estrogen andheregulin)-stimulated spheroid growth in vitro. FIG. 4A shows the effectof MM-111, tamoxifen, or MM-111 and tamoxifen. FIG. 4B shows the effectof trastuzumab, tamoxifen, or trastuzumab and tamoxifen. FIG. 4C showsthe effect of MM-111, fulvestrant (FVT), or MM-111 and fulvestrant. FIG.4D shows the effect of trastuzumab, fulvestrant, or trastuzumab andfulvestrant. FIG. 4E shows the effect of MM-111, trastuzumab, or MM-111and trastuzumab. FIG. 4F shows the effect of MM-111, trastuzumab, andtamoxifen combined compared to that of any of the double combinations.FIG. 4G shows the effect of MM-111, trastuzumab, and fulvestrantcombined compared to that of any of the double combinations. The x-axesare a log scale of each drug concentration for each experimentalcondition in nM and the y axis is spheroid size as % of control spheroidsize.

FIG. 5A is a graph summarizing the effect of MM-111, trastuzumab, andtamoxifen combined on BT474M3 spheroids as compared to that of any ofthe double combinations. The y-axis is % inhibition of spheroid sizenormalized to stimulated control.

FIG. 5B is a graph summarizing the effect of MM-111, trastuzumab, andfulvestrant combined compared to that of any of the double combinationsat inhibiting single ligand (estrogen or heregulin) or dual-ligand(estrogen and heregulin)-stimulated spheroid growth in vitro. The y-axisis % inhibition of spheroid size normalized to stimulated control.

FIG. 6 is a graph showing that the combination of MM-111 and lapatinibinhibits tumor growth in vivo. The x-axis shows the time post tumorimplant in days and the y-axis shows tumor volume in mm³. Mice weretreated with inhibitors on day 7 post tumor implant.

FIGS. 7A and 7B are graphs showing the ability of lapatinib to inhibitErbB3 and AKT activation in heregulin-stimulated cells. FIG. 7A is agraph comparing computer-generated dose-response curves to experimentalresults in heregulin-stimulated BT474-M3 cells. FIG. 7B is a graphshowing lapatinib inhibition (IC50) of ErbB3 and AKT activation inheregulin-stimulated and unstimulated cells following a 1-hourincubation with inhibitor.

FIGS. 8A-8H are a series of graphs showing MM-111 or lapatinibinhibition of ErbB3 (FIGS. 8A-8D) or AKT (FIGS. 8E-8H) activation inheregulin-stimulated cells incubated with inhibitor for 15 minutes, 1hour, 4 hours, and 24 hours.

FIGS. 8I and 8J are graphs showing a comparison of IC50 for MM-111 andlapatinib at 1 hour and 24 hours for both BT474M3 cells and ZR75-30cells.

FIG. 9 is a graph showing the effect of MM-111 and lapatinib combinationtreatment on AKT activation in heregulin-stimulated BT474-M3 cells.

FIG. 10 is a graph showing the effect of lapatinib on cell viability asa measure of proliferation of unstimulated and heregulin-stimulatedBT474-M3 cells.

FIG. 11 is a graph showing the effect of MM-111, lapatinib, or thecombination on BT474-M3 cell apoptosis. The number of dead cells, cellsin late apoptosis, early apoptosis, and live cells was quantitated.

FIGS. 12A-12C are three graphs showing that MM-111 combines positivelywith anti-estrogen drugs and lapatinib in inhibiting dual ligand(estrogen (E2) and heregulin (HRG))-stimulated spheroid growth in vitro.FIG. 12A shows the effect of lapatinib alone or the combination oflapatinib and fulvestrant (FVT). FIG. 12B shows the effect of lapatinibalone or the combination of lapatinib and MM-111. FIG. 12C shows theeffect of lapatinib alone, the combination of MM-111 and fulvestrant, orthe triple combination of MM-111, FVT, and lapatinib. Lapatinib is givenin 3.3, 10, or 30 nM doses. The x-axes are a log scale of each of MM-111and/or FVT concentration in nM and the y axis is spheroid size as % ofcontrol (FBS alone) spheroid size.

FIGS. 13A-13D are four graphs showing that MM-111 combines positivelywith the aromatase inhibitor letrozole and the tyrosine kinase inhibitorlapatinib in heregulin (HRG) and androstenedione (A4)-stimulatedBT474-M3-Aro cells that stably express human aromatase, which convertsandrostenedione to estrogen. FIG. 13A shows the effect of letrozole,MM-111, or the combination of letrozole and MM-111. FIG. 13B shows theeffect of lapatinib, MM-111 or the combination of lapatinib and MM-111.FIG. 13C shows the effect of lapatinib, letrozole, or the combination oflapatinib and letrozole. FIG. 13D shows the effect of the dualcombinations of MM-111 and letrozole, MM-111 and lapatinib, lapatiniband letrozole, and the triple combination of MM-111, lapatinib andletrozole. The x-axes are a log scale of MM-111 concentration in nM. Thedrug concentrations are a ratio of 10:20:1 MM-111 to letrozole tolapatinib. The y axis is spheroid size as % of control spheroid size.

FIGS. 14A-B are two graphs showing that MM-111 combines positively withthe PI3K/mTOR inhibitor BEZ235 and trastuzumab in unstimulated (FIG.14A) and heregulin-stimulated (FIG. 14B) NCI-N87 cells in vitro. Cellswere plated and either untreated (Control, open circle), treated withBEZ235 alone (open diamond), or treated with BEZ235 (5 nM FIG. 14A, 20nM FIG. 14B) and either trastuzumab (closed circle), MM-111 (closedsquare), or the combination of MM-111 and trastuzumab (closed diamond).Closed circles (trastuzumab) may appear as squares or rectangles ontrastuzumab curves when they comprise error bars. The x-axes are a logscale of the concentrations of each of MM-111 and/or trastuzumab.

FIGS. 15A-B are two graphs showing that MM-111 combines positively withthe mTOR inhibitor everolimus and trastuzumab in unstimulated (FIG. 15A)and heregulin-stimulated (FIG. 15B) NCI-N87 cells in vitro. Cells wereplated and either untreated (Control, open circle), treated witheverolimus alone (open diamond), or treated with everolimus (1 nM FIG.15A, 5 nM FIG. 15B) and either trastuzumab (closed circle), MM-111(closed square), or the combination of MM-111 and trastuzumab (closeddiamond). Closed circles may appear as squares or rectangles ontrastuzumab curves when they comprise error bars. The x-axes are a logscale of the concentrations of each of MM-111 and/or trastuzumab.

FIGS. 16A-B are two graphs showing that MM-111 combines positively withthe MEK inhibitor GSK1120212 (trametinib) in a mouse xenograft study.Phospho-ERK was measured as a function of ERK activity via a Luminex®immunosandwich assay at 4 hours after the initial treatment (FIG. 16A)and 24 hours after the initial treatment (FIG. 16B) as a function of ERKactivity via a Luminex immunosandwich assay. The x-axes show type oftreatment and the y-axes show mean fluorescent intensity.

FIGS. 17A-B are two graphs showing that MM-111 combines positively withT-DM1 (ado-trastuzumab emtansine) in both the absence (FIG. 17A) andpresence (FIG. 17B) of endogenous heregulin. The x-axes show a log scaleof T-DM1 concentration and the y-axes show viability as % of control.

FIGS. 18A-D are a series of 3D graphs showing that MM-111 combinespositively with TDM-1 (ado-trastuzumab emtansine) in the presence ofexogenous heregulin. NCI-N87 (FIGS. 18A and 18B) or BT-474-M3 (FIGS. 18Cand 18D) cells were incubated with a dose range of both T-DM1 andheregulin, either without MM-111 (FIGS. 18A and 18C) or with MM-111(FIGS. 18B and 18D). The x-axes are the concentration of T-DM1, they-axes are cell viability as % of untreated cells, and the z-axes areheregulin concentration. The data presented in these graphs is also setforth below in Tables 1-4.

DETAILED DESCRIPTION

As herein provided, bispecific anti-ErbB2/anti-ErbB3 antibodies (e.g.,MM-111) are co-administered with one or more additional therapeuticagents (e.g. an aromatase inhibitor or tyrosine kinase inhibitor), toprovide effective treatment to human patients having a cancer. Suchco-administrations beneficially have an additive or superadditive effecton suppressing tumor cell growth, which effect on suppressing tumor cellgrowth is measured, e.g., in a mouse xenograft model e.g., using BT-474or NCI-N87 cells.

The term “anti-ErbB3 agent” refers to any therapeutic agent that bindsto ErbB3 or binds to an ErbB3-specific ligand or blocks the expressionof ErbB3, and thereby inhibits the activity of cellular signalingmediated by ErbB3. Non-limiting examples of types of anti-ErbB3 agentsinclude antibodies, bispecific antibodies, ligand analogs, soluble formsof ErbB3 or the ErbB3 ectodomain, ErbB3 specific RNAi molecules, andsimilar biologic agents.

The term “antibody” describes a polypeptide comprising at least oneantibody-derived antigen binding site (e.g., V_(H)/V_(L) region or Fv,or complementarity determining region—CDR) that specifically binds to aspecific antigen, e.g., ErbB3. “Antibodies” include whole antibodies andany antigen binding fragment, e.g., Fab or Fv, or a single chainfragment (e.g., scFv), as well as bispecific antibodies and similarengineered variants, human antibodies, humanized antibodies, chimericantibodies Fabs, Fab′2s, ScFvs, SMIPs, Affibodies®, nanobodies, ordomain antibodies, and may be of any of the following isotypes: IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE. The antibodymay be a naturally occurring antibody or may be an antibody that hasbeen altered (e.g., by mutation, deletion, substitution, conjugation toa non-antibody moiety). For example, an antibody may include one or morevariant amino acids (compared to a naturally occurring antibody) whichchange a property (e.g., a functional property) of the antibody. Forexample, numerous such alterations are known in the art which affect,e.g., half-life, effector function, and/or immune responses to theantibody in a patient. The term “antibody” thus includes wholeantibodies and any antigen binding fragment (i.e., “antigen-bindingportion,” e.g., Fabs) or single chains thereof (e.g., scFvs) as well asbispecific antibodies and similar engineered variants, provided thatthey retain the binding specificity of an antibody.

An “anti-ErbB3 antibody” is an antibody that immunospecifically binds tothe ectodomain of ErbB3 and an “anti-ErbB2 antibody” is an antibody thatimmunospecifically binds to the ectodomain of ErbB2. The antibody may bean isolated antibody. Such binding to ErbB3 or ErB2 exhibits a Kd with avalue of no greater than 50 nM as measured by a surface plasmonresonance assay or a cell binding assay. Exemplary anti-ErbB3 antibodiesinhibit EGF-like ligand mediated phosphorylation of ErbB3, e.g.,anti-ErbB2 antibodies that inhibit the binding of heregulin toErbB2/ErbB3 heterodimers. EGF-like ligands include EGF, TGFα,betacellulin, heparin-binding epidermal growth factor, biregulin,epigen, epiregulin, and amphiregulin, which typically bind to ErbB1 andinduce heterodimerization of ErbB1 with ErbB3.

The term “bispecific antibody” as used herein refers to a proteincomprising two antigen-binding sites, a first binding site exhibitingimmunospecific binding to a first antigen or epitope and a secondbinding site exhibiting immunospecific binding to a second antigen orepitope distinct from the first. An “anti-ErbB2/anti-ErbB3 bispecificantibody” is an antibody that comprises two binding sites, one thatimmunospecifically binds to the ectodomain of ErbB3 and another thatimmunospecifically binds to the ectodomain of ErbB2. An exemplarybispecific ErbB3, ErbB2 antibody is an antibody comprising SEQ ID NO:1.

An “anti-estrogen agent” as used herein refers to an agent that preventsor reduces production of estrogen or prevents or reduces signalingmediated by estrogen receptors. Anti-estrogen agents include but are notlimited to estrogen receptor antagonists and aromatase inhibitors.Estrogen receptor antagonists include but are not limited to raloxifene,fulvestrant, tamoxifen, afimoxifene (4-hydoroxytamoxifen), arzoxifene,toremifene, and lasofoxone. Preferably, the estrogen receptor antagonistis tamoxifen or fulvestrant. Aromatase inhibitors work by blocking thesynthesis of estrogen in an animal (e.g., a mouse or a human). Thislowers estrogen levels in the animal and thereby inhibits the growth ofestrogen-driven cancers. Examples of aromatase inhibitors include butare not limited to exemestane, anastrozole, letrozole,aminoglutethimide, testolactone, vorozole, formestane, and fadrozole.Preferably, the aromatase inhibitor is exemestane or letrozole.

By “cancer” is meant any condition characterized by abnormal,unregulated, malignant cell growth.

By “malignant tumor” is meant any cancer that takes the form of a tumor.

The term “effective amount” refers to an amount of a drug effective toachieve a desired effect, e.g., to ameliorate disease in a subject.Where the disease is a cancer, the effective amount of the drug mayinhibit (e.g., slow to some extent, inhibit or stop) one or more of thefollowing characteristics: cancer cell growth, cancer cellproliferation, cancer cell motility, cancer cell infiltration intoperipheral organs, tumor metastasis, and tumor growth. Where the diseaseis a cancer, the effective amount of the drug may alternately do one ormore of the following when administered to a subject: slow or stop tumorgrowth, reduce tumor size (e.g., volume or mass); relieve to some extentone or more of the symptoms associated with the cancer, extendprogression free survival, result in an objective response (including apartial response or a complete response), and increase overall survivaltime. To the extent the drug may prevent growth and/or kill existingcancer cells, it is cytostatic and/or cytotoxic.

A “mouse effective amount” refers to an amount of a drug effective toachieve a desired effect when the subject is a mouse.

A “human effective amount” refers to an amount of a drug effective toachieve a desired effect when the subject is a human patient.

The terms “combination therapy,” “concomitant use,” “co-administration,”co-administering,” “co-administered,” and the like, refer to theadministration of at least two therapeutic agents to a subject eithersimultaneously or within a time period during which the effects of theearlier-administered therapeutic agent are still operative in thesubject when a later-administered therapeutic agent is administered.

A “receptor tyrosine kinase inhibitor” as used herein refers to a memberof a class of drugs that specifically inhibit receptor tyrosine kinasesand thus reduce or eliminate the activation of various signaltransduction pathways. Receptor tyrosine kinase inhibitors useful forthe treatment of cancer as disclosed herein include but are not limitedto the small molecule inhibitors erlotinib, afatinib, dasatinib,gefitinib, imatinib, pazopinib, lapatinib, sunitinib, nilotinib andsorafenib. Receptor tyrosine kinase inhbitors also includeantibody-based therapeutics such as cetuximab, panitumumab, zalutumumab,nimotuzumab, and matuzumab). Preferably, the receptor tyrosine kinaseinhibitor is lapatinib.

“Dosage” or “dosing regimen” refers to parameters for administering adrug in defined quantities per unit time (e.g., per hour, per day, perweek, per month, etc.) to a patient. Such parameters include, e.g., thesize of each dose. Such parameters also include the configuration ofeach dose, which may be administered as one or more units, e.g., takenat a single administration, e.g., orally (e.g., as one, two, three ormore pills, capsules, etc.) or injected (e.g., as a bolus). Dosage sizesmay also relate to doses that are administered continuously (e.g., as anintravenous infusion over a period of minutes or hours). Such parametersfurther include frequency of administration of separate doses, whichfrequency may change over time. A “dosing cycle” or “dosing interval” isthe period of time that comprises one cycle of treatment (e.g., 21 daysor 28 days) for a dosing regimen.

“Dose” refers to an amount of a drug given in a single administration.

In combination, the components of the combination of theanti-ErbB2/ErbB3 antibody have an additive or superadditive effect onsuppressing tumor cell growth, as compared to monotherapy with theanti-ErbB2/ErbB3 antibody or treatment with the other agents in theabsence of antibody therapy. By “additive” is meant a result that isgreater in extent (e.g., in the degree of reduction of tumor mitoticindex or of tumor growth or in the degree of tumor shrinkage or thefrequency and/or duration of symptom-free or symptom-reduced periods)than the best separate result achieved by monotherapy with eachindividual component, while “superadditive” is used to indicate a resultthat exceeds in extent the sum of such separate results. In oneembodiment, the additive effect is measured as slowing or stopping oftumor growth or tumor cell proliferation. The additive effect can alsobe measured as, e.g., reduction in size of a tumor, reduction of tumormitotic index, reduction in number of metastatic lesions over time,increase in overall response rate, or increase in median or overallsurvival.

One non-limiting example of a measure by which effectiveness of atherapeutic treatment can be quantified is by calculating the log 10cell kill, which is determined according to the following equation:

log 10cell kill=TC(days)/3.32×Td

in which T C represents the delay in growth of the cells, which is theaverage time, in days, for the tumors of the treated group (T) and thetumors of the control group (C) to have reached a predetermined value (1g, or 10 mL, for example), and Td represents the time, in days necessaryfor the volume of the tumor to double in the control animals Whenapplying this measure, a product is considered to be active if log 10cell kill is greater than or equal to 0.7 and a product is considered tobe very active if log 10 cell kill is greater than 2.8. Using thismeasure, a combination, used at its own maximum tolerated dose, in whicheach of the constituents is present at a dose generally less than orequal to its maximum tolerated dose, exhibits therapeutic synergy whenthe log 10 cell kill is greater than the value of the log 10 cell killof the best constituent when it is administered alone. In an exemplarycase, the log 10 cell kill of the combination exceeds the value of thelog 10 cell kill of the best constituent of the combination by at least0.1 log cell kill, at least 0.5 log cell kill, or at least 1.0 log cellkill.

Preferred cancer cells of cell lines are cells of ErbB2 expressing celllines such as ErbB2 overexpressing cell lines, e.g., BT474-M3 (ATCC® #CRL-HTB-20™, derived from breast ductal carcinoma cells), BT474-M3-Aro(BT474-M3 cells that stably express human aromatase), ZR75-30 (ATCC® #CRL1504™, derived from breast ductal carcinoma cells), SKOV-3 (ATCC® #HTB-77™, derived from metastatic ovarian adenocarcinoma cells), MCF7(ATCC® # HTB-22™) clone 18, MDA-MB-453 (ATCC® # HTB-131™, derived frombreast carcinoma cells), SK-BR-3 (ATCC® # HTB-30™, derived from breastadenocarcinoma cells), and NCI-N87 (ATCC® # CRL-5822™, derived fromgastric carcinoma cells).

Cancers may include, for example, solid tumors such as: sarcomas (e.g.,clear cell sarcoma), carcinomas (e.g., renal cell carcinoma), andlymphomas; tumors of the breast, colon, rectum, lung, oropharynx,hypopharynx, esophagus, gastric esophageal junction, stomach, pancreas,liver, bilecyst, bile duct, small intestine, urinary system (includingthe kidney, bladder, and epithelium of the urinary tract), femalegenital system (including the uterine neck, uterus, ovary, chorioma, andgestational trophoblast), male genital system (including the prostate,seminal vesicle, and testicles), endocrine glands (including the thyroidgland, adrenal gland, and pituitary body), skin (including angioma,melanoma, sarcoma originating from bone or soft tissue, and Kaposi'ssarcoma), brain and meninges (including astrocytoma, neuroastrocytoma,spongioblastoma, retinoblastoma, neuroma, neuroblastoma, neurinoma andneuroblastoma), nerves, and eyes.

A cancer may be an estrogen receptor positive (ER+) cancer. Such cancersexemplify candidates for therapy regimens that include anti-estrogenagents. Such cancers may include but are not limited to certain breast,ovarian, uterine, endometrial, lung, bone, brain, bladder, liver andurogenital cancers.

A cancer may be an ErbB2 gene-amplified cancer and/or anErbB2-expressing or overexpressing cancer. ErbB2, also known as HER2 orNeu, is a cell surface transmembrane receptor protein that generatesintracellular signals (e.g., upon ligand activation) via itsintracellular tyrosine kinase activity. In excess, such signals canpromote oncogenesis e.g., by triggering cell division. The ErbB2 gene isamplified and/or overexpressed in many types of human malignancies,including but not limited to breast, ovarian, endometrial, pancreatic,colorectal, prostate, salivary gland, kidney, and lung. ErbB2overexpressing cancers are designated a HER2⁺⁺⁺ or HER2⁺⁺ depending onthe level of ErbB2 overexpression, with HER2⁺⁺⁺ indicating the highestlevels of HER2 expression. HER2⁺⁺⁺ and HER2⁺⁺ status are typicallydetermined by an immunoassay such as immunohistochemistry, e.g.,Herceptest®. ErbB2 gene amplification may be determined by, e.g., FISH(fluorescence in situ hybridization), with HER2-amplified cancer cellsbeing those that have more than two HER2 gene copies beingHER2-amplified, and cells and/or tumors comprising HER2-amplified cancercells being referred to as “FISH positive.”

A number of bispecific anti-ErbB2, antiErbB3 antibodies that are scFvHSA conjugates are described in co-pending US patent publication No.2011-0059076, and PCT publication Nos. WO2009/126920 and WO 2010/059315,MM-111 (also referred to as B2B3-1) and other bispecificanti-ErbB2/antiErbB3 antibodies that are scFv HSA conjugates and thatare suitable for use in the methods and compositions provided herein,including the components of A5-HSA-ML3.9, ML3.9-HSA-A5, A5-HSA-B1D2,B1D2-HSA-A5, B12-HSA-B1D2, B1D2-HSA-B12, A5-HSA-F5B6H2, F5B6H2-HSA-A5,H3-HSA-F5B6H2, F5B6H2-HSA-H3, F4-HSA-F5B6H2, F5B6H2-HSA-F4, B1D2-HSA-H3,and H3-HSA-B1D2. Other suitable bispecific anti-ErbB2/antiErbB3antibodies are disclosed and claimed in U.S. Pat. Nos. 7,332,580 and7,332,585. MM-111 is currently undergoing clinical trials, including anopen-label Phase 1/2 and pharmacologic study of MM-111 in patients withadvanced, refractory HER2 positive cancers, an open-label Phase 1/2trial of MM-111 in combination with trastuzumab (Herceptin®) in patientswith advanced HER2 positive breast cancer, an open label, Phase 1/2 andpharmacologic study of MM-111 with five different combinationtreatments: MM-111 in combination with cisplatin, capecitabine, andtrastuzumab, MM-111 in combination with lapatinib and trastuzumab, andMM-111 in combination with paclitaxel and trastuzumab, MM-111 incombination with lapatinib, paclitaxel and trastuzumab, and MM-111 incombination with docetaxel and trastuzumab, and an open label, Phase 2study of MM-111 and paclitaxel with or without trastuzumab in patientswith HER-2 expressing carcinomas of the distal esophagus,gastroesophageal junction and stomach.

A bispecific anti-ErbB2/anti-ErbB3 antibody (e.g., MM-111) can beco-administered with other therapeutic agents, (e.g, an anti-estrogenreceptor agent or a receptor tyrosine kinase inhibitor) prior to (e.g.,neoadjuvant therapy), concurrent with, or following (e.g., adjuvanttherapy) radiotherapy of, or surgical intervention to remove, amalignant tumor.

Additional therapeutic agents suitable for combination withanti-ErbB2/anti-ErbB3 antibodies may further include: 1) antibody EGFRinhibitors (e.g. MM-151, Sym004, cetuximab, panitumumab, zalutumumab,nimotuzumab, and matuzumab), additional small molecule tyrosine kinaseinhibitors such as PKI-166, PD-158780, EKB-569 (pelitinib), tyrphostinAG-1478, and pan-HER kinase inhibitors (e.g. CI-1033 (canertinib, PD183805), AC480/BMS-599626, HM781-36B, AZD8931 (sapitinib) and PF299804(dacomitinib)); 2) microtubule stabilizing agents (e.g. laulimalide,epothilone A, epothilone B, discodermolide, eleutherobin, sarcodictyinA, sarcodictyin B, paclitaxel, nab-paclitaxel or docetaxel);antimetabolites such as 5-fluorouracil (5-FU) and capecitabine; 3)platinum-based therapeutics such as oxaliplatin, carboplatin andcisplatin; 4) mTOR inhibitors such as BEZ235 (a dual PI3K/mTORinhibitor), AZD8055, everolimus, temsirolimus, siroplimus/rapamycin, andridaforolimus; and 5) additional anti-ErbB2 therapeutic agents, such astrastuzumab, pertuzumab, and T-DM1 (ado-trastuzumab emtansine).Additional examples of therapeutic agents suitable for combination withanti-ErbB2/anti-ErbB3 antibodies may be found in Table 5 and theAppendix below.

MM-111 is suitable for both large scale production and systemic therapy.MM-111 binds to ErbB2/ErbB3 heterodimers and forms a trimeric complexwith ErbB2 and ErbB3, effectively inhibiting ErbB3 signaling. Theantitumor activity of MM-111 requires the presence of both ErbB2 andErbB3, but is particularly dependent on ErbB2 expression. The affinityof its ErbB2 antigen-binding site is about 30 times higher than theaffinity of its ErbB3 antigen-binding site, but the ErbB2antigen-binding site does not by itself inhibit ErbB2 activity whenbound to ErbB2. The strong binding of MM-111 to ErbB2 places the ErbB3antigen-binding site in close proximity to bound ErbB2/ErbB3heterodimer, resulting in an avidity effect that potentiates the bindingof the ErbB3 antigen-binding site to the heterodimer ErbB3, whereby abiological effect is produced. MM-111 is administered to human subjects(patients) at an interval measured in days, as a single loading dose ofat least 20 mg/kg of MM-111 followed by at least seven day intervals(e.g., every 2 weeks) by at least one administration of a singlemaintenance dose of MM-111, where the maintenance dose is generallysmaller than the loading dose, e.g., at least 5 mg/kg less than theloading dose.

Examples

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

MM-111 in Combination with Anti-Estrogen Therapeutics

Methods: Spheroid In Vitro Tumor Model Assay

BT474-M3 wild type cells (2000 cells/well) are plated in Ultra LowCluster 96-well plate (Costar). After overnight incubation, indicatedtreatments are introduced to the plate. Cells are continued to culturefor six days. Spheroids are then examined by Nikon microscope andanalyzed by MetaMorph Image Analysis Software (Molecular Devices). Thespheroid size from cells cultured in medium containing 10% FBS is set ascontrol.

Xenograft Model

BT474-M3 cells (2×10⁷ cells per mice) are inoculated subcutaneously intoNu/Nu immunodeficient mice, which are implanted with an estrogen pellet(0.72 mg; 60-day release) one day before the experiment. Tumors aremeasured after seven days and mice are randomized into four groups:those treated with placebo, MM-111 (60 mg/kg, Q7D), 4-hydroxytamoxifen(5 mg; 60-day release pellet), and combination of MM-111 and4-hydroxytamoxifen, respectively. Tumors are measured every three daysand the experiment is ended at day 32.

Example 1: MM-111 and Tamoxifen Combination Therapy Inhibits TumorGrowth In Vivo

In order to compare the effect of MM-111 and tamoxifen combinationtherapy on tumor growth in vivo, estrogen stimulated mice were preparedin the xenograft model using the methods described above or minorvariations thereof. Mice were inoculated with tumor forming BT474-M3cells and on day 7 given a placebo (vehicle control), MM-111, tamoxifen,or a combination of MM-111 and tamoxifen and tumor growth was measuredover time. As shown in FIG. 1, this in vivo BT474-M3 xenograft modelshowed resistance to tamoxifen treatment but when mice were given acombination of MM-111 and tamoxifen the combination treatment inhibitedtumor growth to a significantly greater extent. Statistical significance(p<0.05) was observed for the combination group from day 28 onward whencompared to vehicle control, from day 21 onward when compared to MM-111and from day 25 onward when compared to tamoxifen.

Example 2: MM-111 Combines Positively with Anti-Estrogen Drugs inInhibiting Estrogen-Stimulated Spheroid Growth

Multicellular spheroids are used to simulate the growth andmicroenvironmental conditions of tumors in vitro. To further investigatethe ability of MM-111 to inhibit cell growth when in combination withanti-estrogen drugs, spheroids of BT474-M3 cells were prepared using themethods described above or minor variations thereof and treated with anErbB2-binding therapeutic and/or an anti-estrogen therapeutic. Spheroidsof estrogen-stimulated cells were treated with a dose range of MM-111,tamoxifen, or the combination of MM-111 and tamoxifen (FIG. 2A);trastuzumab, tamoxifen or the combination of trastuzumab and tamoxifen(FIG. 2B); MM-111, fulvestrant, or the combination of MM-111 andfulvestrant (FIG. 2C); trastuzumab, fulvestrant, or the combination oftrastuzumab and fulvestrant (FIG. 2D); or MM-111, trastuzumab, or thecombination of MM-111 and trastuzumab (FIG. 2E). When used as singleagent alone, MM-111, trastuzumab, fulvestrant and tamoxifen showedinhibitory effects on spheroid growth in the estrogen-stimulatedBT474-M3 spheroid assay. The combination of tamoxifen or fulvestrantwith MM-111 (FIGS. 2A and 2C, respectively) or trastuzumab (FIGS. 2B and2D, respectively) increased the degree of growth inhibition, as did thecombination of MM-111 and trastuzumab (FIG. 2E). The inhibitory effectswere increased still further when estrogen-stimulated spheroids weretreated with the triple combination of MM-111, trastuzumab, andtamoxifen (FIG. 2F) or MM-111, trastuzumab, and fulvestrant (FIG. 2G) ascompared to the double combinations of drugs.

Example 3: MM-111 Combines Positively with Anti-Estrogen Drugs inInhibiting Heregulin-Stimulated Spheroid Growth

To further investigate the ability of MM-111 to inhibit cell growth whenin combination with anti-estrogen drugs, spheroids of heregulin(HRG)-stimulated BT474-M3 cells were prepared using the methodsdescribed above or minor variations thereof and treated with a doserange of MM-111, tamoxifen, or the combination of MM-111 and tamoxifen(FIG. 3A); trastuzumab, tamoxifen or the combination of trastuzumab andtamoxifen (FIG. 3B); MM-111, fulvestrant, or the combination of MM-111and fulvestrant (FIG. 3C); trastuzumab, fulvestrant, or the combinationof trastuzumab and fulvestrant (FIG. 3D); or MM-111, trastuzumab, or thecombination of MM-111 and trastuzumab (FIG. 3E). MM-111 inhibitedheregulin-induced spheroid growth but tamoxifen (FIG. 3A), trastuzumab(FIG. 3B), and fulvestrant (FIG. 3C) did not inhibit heregulinstimulated spheroid growth. No significant combinational effect wasobserved when MM-111 was used with tamoxifen (FIG. 3A) or fulvestrant(FIG. 3C). The combination of trastuzumab and either tamoxifen (FIG. 3B)or fulvestrant (FIG. 3D) failed to show inhibitory activitysignificantly greater than either drug alone. As shown in FIG. 3E,MM-111 but not trastuzumab showed inhibitory activity inheregulin-stimulated spheroid growth. Improved inhibitory effects wereobserved when both drugs were combined. In comparison to the doublecombination of either MM-111 or trastuzumab with tamoxifen orfulvestrant, the triple combination of MM-111, trastuzumab and eithertamoxifen (FIG. 3F) or fulvestrant (FIG. 3G) showed similar inhibitoryeffects as those of MM-111 and trastuzumab in combination (FIG. 3E) onheregulin-stimulated spheroid growth.

Example 4: MM-111 Combines Positively with Anti-Estrogen Drugs inInhibiting Dual Ligand (Estrogen and Heregulin)-Stimulated SpheroidGrowth

Dual ligand (estrogen and heregulin) stimulated spheroids were treatedwith a dose range of tamoxifen, MM-111 or the combination of MM-111 andtamoxifen (FIG. 4A) or trastuzumab, tamoxifen or the combination oftrastuzumab and tamoxifen (FIG. 4B). While MM-111 and trastuzumab eachinhibited spheroid growth (FIG. 4A) the combination of MM-111 andtamoxifen showed greater inhibitory effects than either drug alone. Incontrast, trastuzumab alone had no significant inhibitory effects andthe combination of trastuzumab and tamoxifen showed similar effects totamoxifen alone.

Dual ligand stimulated spheroids were then treated with a dose range offulvestrant, MM-111 or the combination of MM-111 and fulvestrant (FIG.4C) or fulvestrant, trastuzumab, or a combination of fulvestrant ortrastuzumab (FIG. 4D). Again, while MM-111 and fulvestrant eachseparately inhibited spheroid growth the combination of MM-111 andfulvestrant showed greater inhibitory effects than either drug alone(FIG. 4C). Trastuzumab alone had no significant inhibitory effects andthe combination of trastuzumab and fulvestrant showed similar effects totamoxifen alone (FIG. 4D).

Dual ligand stimulated spheroids were then treated with MM-111,trastuzumab, or a combination of MM-111 and trastuzumab. MM-111 showedgreater inhibitory effects than trastuzumab in dual ligand-stimulatedspheroid growth. Enhanced inhibitory effects were observed when bothdrugs were combined (FIG. 4E).

In comparison to the double combination of MM-111 or trastuzumab withtamoxifen or fulvestrant, the triple combination of MM-111, trastuzumaband either tamoxifen (FIG. 4F) or fulvestrant (FIG. 4G) showed similarinhibitory effects to those of MM-111 and trastuzumab in combination(FIG. 4E) on estrogen- and heregulin- (dual ligand) stimulated spheroidgrowth.

The data in the preceding Examples demonstrate that combinationtherapies comprising MM-111 and an anti-estrogen therapeutic are moreeffective than each of these therapies alone. The percent of spheroidgrowth inhibition induced by each treatment under estrogen or heregulinstimulation is summarized in FIGS. 5A-B and Table 1. MM-111 was requiredfor inhibition of spheroids stimulated with heregulin. For eachstimulated condition tested, the triple combination resulted in thegreatest inhibition of spheroid growth, providing a percent inhibitionranging from about 70% to about 90%.

TABLE 1 Percent inhibitor induced maximal spheroid growth inhibitionMM-111 + MM-111 + Trastuzumab + Triple Trastuzumab anti-estrogenanti-estrogen combination Tamoxifen combination E2 54% 49% 55% 73% HRG65% 43% 0% 71% E2 + HRG 46% 43% 36% 79% Fulvestrant combination E2 54%49% 55% 77% HRG 64% 34% 4% 71% E2 + HRG 46% 57% 47% 88%The percent of spheroid growth inhibition (normalized to untreated,stimulated control) was determined for 1 μM doses of inhibitortreatment.

The combination of MM-111 and tamoxifen resulted in potent inhibition oftumor growth in vivo. Taken together, these data demonstrate that thecombination of MM-111 and anti-estrogen therapies results in potentanti-tumor effects in vitro and in vivo.

MM-111 in Combination with Lapatinib

Methods Computational Modeling

A computational model of HRG-induced phospho-ErbB3 signaling, as well asa model of lapatinib, was used as previously described (Schoeberl, et al2009).

Cell Signaling Assay

Serum-starved cells are pre-incubated with serial dilutions of MM-111,lapatinib or combinations at doses and treatment times indicated,followed by stimulation with 5 nM heregulin 1-β (R&D Systems,Minneapolis, Minn.) for 10 minutes. Cell lysates are probed forphospho-ErbB3 (pErbB3), and phospho-AKT (pAKT) by ELISA as describedpreviously (Schoeberl et al, 2009). Inhibitor IC₅₀ values are calculatedby fitting dose-response data to a 4-parameter sigmoidal curve (GraphPadPrism®, GraphPad Software, Inc., La Jolla, Calif.).

Cell Proliferation Assay

Cells (8,000/well) are seeded into 96-well plates and incubatedovernight. Inhibitor is added at doses indicated and cells are treatedfor 24 hours. For experiments with ligand stimulation, cells areserum-starved overnight prior to addition of inhibitor and 2 nMheregulin 1-□(R&D Systems, Minneapolis, Minn.) is added 1 hourpost-inhibitor treatment in media containing 5% FBS. Numbers of viablecells are measured as an indicator of cell proliferation using theCellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison,Wis.).

Apoptosis Assay

BT474-M3 cells (2000 cells/well) are plated in Ultra Low Cluster 96-wellplate (Costar®, Corning, N.Y.). After overnight incubation, spheroidsare treated with inhibitor at concentrations indicated for 72 hours.Spheroids are then trypsinized and combined with floating cells. Cellsare washed twice with cold PBS and suspended in binding buffer (0.01 MHEPES, pH 7.4; 0.14 M NaCl; 2.5 mM CaCl₂). Cells are then stained withFITC-conjugated Annexin V and PI. Apoptotic cells are quantified on aFACSCalibur™ FACS machine.

Xenograft Efficacy Studies

Tumor xenografts are established by subcutaneous injection of BT474-M3cells into the flank of 5-6 weeks old female athymic nude mice (nu/nu;Charles River Labs, Wilmington, Mass.). Mice receive a subcutaneous 60day, slow-release estrogen implant in the opposite flank (0.72 mgpellet; Innovation Research of America, Sarasota, Fla.) 24 hours priorto the injection of cells. Once tumors reach a mean volume of 150-500mm³, mice are randomized into groups of 8 or 10 and dosed byintraperitoneal injection once every three days with vehicle, MM-111 orlapatinib. For lapatinib combination studies, MM-111 is given once everyseven days and lapatinib daily by gavage at doses indicated.

Aromatase-Overexpressing BT474-M3 Cells and Proliferation Assay

BT474-M3 cells were transfected with PS100010 vector containing humanaromatase (gene accession No: NM_000103.2). Cells with stable expressionof aromatase (BT474-M3-Aro) were obtained after selection with 400 μg/mlgeneticin. For cell proliferation assay, BT474-M3-Aro cells (5000cells/well) were plated in phenol red-free RPMI-1640 medium containing5% charcoal-stripped FBS into 96-well plate. After overnight incubation,indicated treatments were introduced in the presence of androstenedione(A-4; 200 nM) and heregulin (HRG; 2 nM). After three days of treatment,cell viability was determined by WST-1 (Roche; Cat. #11 644 807 001)according to manufacturer's instruction. Cell viability in the presenceof 5% charcoal-stripped FBS was set as control (100%).

Example 5: The Combination of MM-111 and Lapatinib Inhibits Tumor GrowthIn Vivo

The combination of MM-111 with lapatinib was investigated in vivo in theBT474-M3 breast cancer xenograft model using the methods described aboveor minor variations thereof. MM-111 and lapatinib were each dosed at anoptimal efficacious dose weekly and daily, respectively. The combinationof MM-111 and lapatinib provided more potency compared to either drugalone, reaching statistical significance for MM-111 (p=3.9×10-4) andlapatinib (p=5.1×10-3) on day 13 (FIG. 6). The percent change in tumorvolume from day 40 to day 7 (inoculation) was calculated for each group(FIG. 6). The combination of MM-111 and lapatinib resulted in a percentchange in tumor volume of −69% (about 70%), reflecting tumorregressions, compared to −11% (about 10%) for lapatinib and 14% (about15%) for MM-111.

Example 6: Simulations Predict Lapatinib has Suboptimal Activity inInhibiting Heregulin-Driven pErbB3 and pAKT

A dose range of lapatinib inhibition of pErbB3 activation was predictedusing the computational modeling described above. A dose range oflapatinib was applied to BT474-M3 cells followed by stimulation with 5nM heregulin for 10 min. The amount of pErbB3 was measured by ELISAusing the methods described above or minor variations thereof.Model-generated dose-response curves overlay the experimental data (FIG.7A). A comparison of the inhibitory activity of lapatinib inheregulin-stimulated or unstimulated (basal) cells was performed todemonstrate that heregulin signaling perturbs the activity of lapatinib.Untreated and heregulin-stimulated cells were probed for pErbB3 and pAKTand the IC50 was calculated (FIG. 7B). These data show that lapatinibalone is not an effective inhibitor of heregulin-activated signaling.

Example 7: MM-111 is a More Potent Inhibitor of HRG-Driven ErbB3 and AKTPhosphorylation than Lapatinib

In order to compare the ability of MM-111 and lapatinib to inhibitheregulin-induced ErbB3 activation, BT474-M3, or an additional ErbB2overexpressing breast tumor cell line, ZR75-30 (ATCC® # CRL-1504™),cells were incubated with serial dilutions of either inhibitor for 15minutes, 1 hour, 4 hours, and 24 hours followed by stimulation with 5 nMheregulin for 10 min. Amounts of pAKT and pErbB3 were measured by ELISAessentially as described. MM-111 potently reduced pErbB3 levels(inhibited ErbB3 phosphorylation) in BT474-M3 (IC₅₀=3 nM) cells (FIG.8A) and ZR75-30 cells (IC₅₀=5 nM) (FIG. 8C). Good reduction by MM-111 ofpAKT levels (inhibition of AKT phosphorylation) in BT474-M3 (IC₅₀=10)(FIG. 8B) and in ZR75-30 cells (IC₅₀=4 nM) (FIG. 8D) was also observed.The ability of MM-111 to inhibit heregulin-induced ErbB3 activation(phosphorylation) was superior to lapatinib by greater than an order ofmagnitude and the relative IC₅₀ for each inhibitor (FIG. 8C) wasconsistent following up to 24 hours incubation with inhibitors,indicating treatment times had little effect on the potency of theinhibitors.

Example 8: The Combination of MM-111 and Lapatinib Potently InhibitspAKT

The effect of MM-111 combined with lapatinib on pAKT inhibition(reduction of pAKT levels) was assessed in heregulin-stimulated BT474-M3cells. Cells were incubated for 2 hours with a dose range of MM-111,lapatinib or their combination and pAKT was measured by ELISA. In thepresence of heregulin, the combination of MM-111 and lapatinib wasextremely effective, inhibiting pAKT well below basal levels attherapeutically relevant concentrations (FIG. 9). Treatment with eitherMM-111 (1 μM) or lapatinib (1 μM) alone resulted in similar levels ofpAKT inhibition (see FIG. 8B) while the combination resulted in about20% more inhibition of pAKT.

Example 9: The Ability of Lapatinib to Inhibit Cell Proliferation isPerturbed Under Heregulin-Stimulated Conditions

The effect of lapatinib on cell proliferation was measured inunstimulated and heregulin-stimulated BT474-M3 cells. Cells grown inserum or in serum plus 2 nM heregulin were treated with lapatinib acrossa dose range for 24 hours. Lapatinib treatment resulted in about a 50%inhibition of unstimulated cells but its effect was reduced to about 23%inhibition in heregulin-stimulated BT474-M3 cells (FIG. 10).

Example 10: Treatment with the Combination of MM-111 and LapatinibResults in Increased Apoptosis

The effect of the MM-111 combination with lapatinib on apoptosis wasassessed in a BT474-M3 spheroid model. Spheroids were prepared using themethods described above or minor variations thereof and treated withMM-111 (100 nM), lapatinib (33 nM), or a combination of 100 nM MM-111and 33 nM lapatinib. Cells were then stained with Annexin V andpropidium iodide (PI) and quantitated using FACS (FIG. 11, Table 2).Cell populations staining positive with Annexin V and PI were quantifiedas late apoptotic, cell populations staining positive with Annexin V butnot PI were quantified as early apoptotic, cell populations stainingpositive for PI but not Annexin V were quantified as dead cells andpopulations of cells not stained with either Annexin V or PI wereconsidered alive and not apoptotic (Table 2). Spheroids that weretreated with both MM-111 and lapatinib had a higher number of totalapoptotic cells (about 46%) compared to those treated with onlylapatinib (about 31%) or only MM-111 (about 20%; FIG. 10).

TABLE 2 Percent cell population after treatment with MM-111, lapatinibor the combination Live cells Early apoptosis Late apoptosis Dead cellsControl 75.2 17.3 7.2 0.42 MM-111 78.9 12.9 7.5 0.74 Lapatinib 67.9 16.814.5 0.73 Combination 52.1 30.0 16.2 1.74

Example 11: MM-111 Combines Positively with Anti-Estrogen Drugs andLapatinib in Inhibiting Dual Ligand (Estrogen and Heregulin)-StimulatedSpheroid Growth

To further investigate the ability of MM-111 to inhibit cell growth whenin combination with both anti-estrogen drugs and tyrosine kinaseinhibitors, spheroids of estrogen and heregulin-stimulated BT474-M3cells were prepared using the methods described above or minorvariations thereof and treated with 3.3 nM, 10 nM, or 30 nM lapatinib,either alone or in combination with a dose range of fulvestrant (FVT)(FIG. 12A); 3.3 nM, 10 nM, or 30 nM lapatinib, either alone or incombination with a dose range of MM-111 (FIG. 12B); or 3.3 nM, 10 nM, or30 nM lapatinib, either alone or in combination with a dose range ofboth MM-111 and fulvestrant (FIG. 12C). In the presence of dual ligandstimulation the combination of lapatinib and FVT did not greatlyincrease inhibition of spheroid growth over lapatinib alone (FIG. 12A).In contrast, the addition of MM-111 greatly increased the sensitivity ofthe spheroids to lapatinib treatment (FIG. 12B), and the triplecombination of lapatinib, FVT and MM-111 showed an even greater increaseof spheroid growth inhibition over lapatinib alone.

Example 12: MM-111 Combines Positively with Anti-Estrogen Drugs inInhibiting Spheroid Growth in BT474-M3 Cells Overexpressing HumanAndrostenedione

Androstenedione is a steroid hormone that is converted to estrogen byaromatase. To further investigate the ability of MM-111 to inhibitspheroid growth, aromatase-expressing cells were treated in the presenceof androstenedione (A4) and heregulin (HRG) with MM-111, letrozole, orthe combination of MM-111 or letrozole (FIG. 13A); MM-111, lapatinib, orthe combination of MM-111 and lapatinib (FIG. 13B); lapatinib,letrozole, or the combination of lapatinib and letrozole (FIG. 13C); andeach of the dual combination plus the triple combination of MM-111,lapatinib, and letrozole (FIG. 13D). In cells treated with A4 and HRG,the letrozole treatment did not result in significant inhibition ofspheroid cell growth as compared to control (untreated) cells, whereascells treated with MM-111 alone or the combination of MM-111 andletrozole inhibited cell proliferation to a similar extent (FIG. 13A).Lapatinib treatment of the cells did not result in growth inhibitionexcept at high concentrations, whereas treatment with MM-111 alone or incombination resulted in similar levels of cell growth inhibition exceptin higher concentrations where the combination showed increasedinhibition of cell growth over either of the single treatments (FIG.13B). Treatment with lapatinib alone, letrozole alone, or thecombination of lapatinib and letrozole did not result in significantcell growth inhibition except at high concentration (FIG. 13C).Similarly, as shown in FIG. 13D, the double combination of lapatinib andletrozole resulted in cell growth inhibition only at high drugconcentration. In contrast the dual combinations of MM-111 and letrozoleor MM-111 and lapatinib both showed an increase in cell growthinhibition as compared to control, and the triple combination of MM-111,lapatinib, and letrozole inhibited cell growth to an even greaterdegree.

Example 13: Amino Acid Sequence of MM-111(SEQ ID NO:1)

QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSASTGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGAASDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFQAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAAALQVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCTDRTCAKWPEWLGVWGQGTLVTVSSGGGGSSGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLG

Dosing and Administration of MM-111 in Combination with One or MoreAdditional Therapeutics Example 14: Mode of Administration of MM-111

MM-111 is prepared as a formulation containing 25 mg/ml MM-111 in asterile aqueous solution comprising 20 mM L-histidine hydrochloride, 150mM sodium chloride, pH 6.5, which is stored at 2-8° C.

MM-111 must be brought to room temperature prior to administration.Containers (e.g., vials) of MM-111 must not be shaken. The appropriatequantity of MM-111 is removed from the container, diluted in 250 mL of0.9% normal saline and administered as an infusion using a low proteinbinding in-line filter (e.g., a 0.22 micrometer filter).

MM-111 is initially administered over about 90 minutes (firstadministration). In the absence of an infusion reaction, subsequentdoses are administered over about 60 minutes.

A patient's body weight at the start of a dosing cycle is used tocalculate the dose used throughout the cycle. Should a patient's bodyweight change by more than 10%, a new total dose is calculated toreflect this change.

Example 15: Dosage and Administration of MM-111

Preferred plasma concentrations of MM-111 achieved during treatment areat least 106 mg/L. It has now been discovered that certain combinationsof dose frequency and dosage will achieve and maintain this plasmaconcentration during the course of treatment in at least half, andpreferably in more than 60%, 70% or 80% of treated patients.

In certain embodiments a higher initial dose (loading dose—LD) is given,followed as defined intervals by at least one maintenance dose (MD).Intervals of dosing in days are typically indicated as QxD, wherein xrepresents an integer, so that a QxD of 7 indicates dosing every 7 days.Table 3A, Table 3B, and Table 3C below show doses and dosing intervalsof the invention. In Table 3A, Table 3B, and Table 3C the indicatedloading doses are optional—initial doses are preferably made at theindicated loading dose (LD), but may (e.g., as directed or at thephysician's discretion) be made at the maintenance dose (MD). Table 3Aprovides a set of exemplary dosing intervals, loading doses andmaintenance doses. Table 3B provides a variation of Table 3A allowingfor dosage variability (indicated as “about”) of up to +/−3 mg/mL. Table3C appears below and provides a more extensive set of exemplary dosingintervals, loading doses and maintenance doses. In each cell of Table3A, Table 3B, and Table 3C, the top figure is the integer x in theinterval QxD (e.g., 18 as the top figure in a cell indicates a dosinginterval of Q18D or every 18 days), the middle figure represents the(optional) loading dose (LD) in mg/kg, and the bottom figure representsthe maintenance dose (MD) in mg/kg. Thus the top cell in Table 3Aindicates a dosing interval (QxD) of once every seven days, a loadingdose (optional) of 25 mg per kg of patient body weight, and amaintenance dose of 20 mg per kg of patient body weight; while the cellfurthest to the right on the top row of Table 3C indicates a dosinginterval (QxD) of once every seven days, a loading dose (optional) of 30mg per kg of patient body weight, and a maintenance dose of 15 mg per kgof patient body weight.

TABLE 3A 7 25 20 7 40 30 14 60 45 14 90 75 21 120 105

TABLE 3B  7 about 25 about 20  7 about 40 about 30 14 about 60 about 4414 about 90 about 75 21 about 120 about 105

TABLE 3C 7 7 7 7 7 7 7 7 7 7 7 7 7 10 15 20 25 30 15 20 25 30 35 20 2530 5 5 5 5 5 10 10 10 10 10 15 15 15 7 7 7 7 7 7 7 7 7 7 7 7 7 35 40 2530 35 40 45 30 35 40 45 50 55 15 15 20 20 20 20 20 25 25 25 25 25 25 7 714 14 14 14 14 14 14 14 14 14 14 60 65 35 40 45 50 55 60 65 70 75 40 4525 25 30 30 30 30 30 30 30 30 30 35 35 14 14 14 14 14 14 14 14 14 14 1414 14 50 55 60 65 70 75 45 50 55 60 65 70 75 35 35 35 35 35 35 40 40 4040 40 40 40 14 14 14 14 14 14 14 14 14 14 14 14 14 50 55 60 65 70 75 5560 65 70 75 60 65 45 45 45 45 45 45 50 50 50 50 50 55 55 14 14 14 14 1414 14 14 21 21 21 21 21 70 75 65 70 75 70 75 75 60 65 70 65 70 55 55 6060 60 65 65 70 55 55 55 60 60 21 21 21 21 21 21 75 70 75 80 85 90 60 6570 75 80 85

Example 16. Dosage and Administration of MM-111 with Lapatinib andTrastuzumab

Treatment for patients with trastuzumab-refractory HER2-overexpressingbreast cancer is a critical unmet need in the field of breast oncology,and novel approaches to address this need are required. Althoughselective tyrosine kinase inhibitors (TKIs) have been highly effectivefor the treatment of certain tyrosine kinase oncogene-driven cancers,their clinical anti-tumor efficacy in the treatment of HER2-drivenbreast cancer has been disappointing despite adequate biodistributionand apparent target inhibition. Two completed phase II trials using themost potent HER2 TKI, lapatinib, have reported response rates of only4%-8% in patients with trastuzumab-refractory HER2-overexpressing breastcancer. It is now known that the effective treatment of HER2+ breastcancer is more complex and resilient than previously thought. Recentevidence has highlighted the role of HER3 and a robust signal bufferingcapacity inherent in the HER2-HER3 tumor driver that protects it againsta two log inhibition of HER2 catalytic activity, placing it beyond thetherapeutic index of even the most potent tyrosine kinase inhibitors(TKIs).

Typically, lapatinib is administered at a dosage of 1000 to 1500 mg in250 mg tablets taken once daily. Lapatinib is often used in combinationwith another cancer medication, capecitabine, which is taken for 14 dayperiods with one week in between.

In order to test whether the full inactivation of the HER2-HER3 drivercan be achieved with much higher TKI dosing at an intermittent dosingschedule is more efficacious than continuous dosing, a modified dosingschedule is used wherein an increased dose of lapatinib is administeredon days 1-5 of a 14 day cycle, said increased dose being a higher dosethan the standard dose of 1000 to 1500 mg/day. In some embodiments, thehigher lapatinib dose is between 2000 and 9000 mg/d. For example, higherlapatinib dose might be 2000, 2250, 3375, 3000, 3250, 3500, 3750, 4000,4250, 4500, 4750, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000,7250, 7500, 7750, 8000, 8250, 8500, 8750, or 9000 mg/day, and so on.

In certain embodiments a loading dose is given on day 1 of the 14-daycycle that is a higher dose than that given on subsequent days, themaintenance dose. For example, a loading dose given on day 1 of the 14day cycle might be 7000 mg/day, followed by a maintenance dose of 3000mg/day. Non-limiting examples of loading dose and maintenance dosecombinations are listed in Table 4 below.

MM-111 is administered as described in Example 15. In some embodimentsthe treatment further comprises trastuzumab. Trastuzumab is typicallygiven with an initial loading dose followed by a maintenance dose. Forexample, trastuzumab may be dosed at a loading dose of 8 mg/kg followedby a maintenance dose of 6 mg/kg every three weeks.

TABLE 4 Exemplary lapatinib dosing schedule: loading dose (top number)and maintenance dose (bottom number) in mg/d 2000 2000 2000 2500 25002500 3000 3000 3000 3000 3000 3500 3500 1000 1500 2000 1000 1500 20001000 1500 2000 2500 3000 1000 1500 3500 3500 3500 4000 4000 4000 40004000 4000 4500 4500 4500 4500 2000 2500 3000 1000 1500 2000 2500 30003500 1000 1500 2000 2500 4500 4500 4500 5000 5000 5000 5000 5000 50005000 5000 5500 5500 3000 3500 4000 1000 1500 2000 2500 3000 3500 40004500 1000 1500 5500 5500 5500 5500 5500 5500 5500 6000 6000 6000 60006000 6000 2000 2500 3000 3500 4000 4500 5000 1000 1500 2000 2500 30003500 6000 6000 6000 6000 7500 7500 7500 7500 7500 7500 7500 7500 75004000 4500 5000 5500 1000 1500 2000 2500 3000 3500 4000 4500 5000 75007500 7500 7500 8000 8000 8000 8000 8000 8000 8000 8000 8000 5500 60006500 7000 1000 1500 2000 2500 3000 3500 4000 4500 5000 8000 8000 80008000 8000 9000 9000 9000 9000 9000 9000 9000 9000 5500 6000 6500 70007500 1000 1500 2000 2500 3000 3500 4000 4500 9000 9000 9000 9000 90009000 9000 9000 5000 5500 6000 6500 7000 7500 8000 8500

Example 17: Dosage and Administration of MM-111 with Cisplatin,Capecitabine, and Trastuzumab

Administration of MM-111 with cisplatin, capecitabine, and trastuzumabis done, for example, by the following method or minor variationsthereof.

Patients are administered therapy on a 21-day treatment cycle. Cisplatinis administered on day 1 of each 21-day cycle by intravenous (i.v.)infusion over two hours, at a dose of 80 mg/m². Capecitabine isadministered orally, twice daily, at a dose of 1000 mg/m². Up to 21-daycycles of cisplatin and capecitabine are administered. Trastuzumab isadministered i.v. at week 1 at an 8 mg/kg loading dose over 90 minutes,followed by a maintenance dose of 6 mg/kg every 21 days over 30-90minutes. MM-111 is administered as described in the above Examples. Forexample, MM-111 is administered i.v. over 90 minutes for the first doseand then weekly over 60 minutes thereafter.

Example 18: Dosage and Administration of MM-111 with Lapatinib andTrastuzumb

Administration of MM-111 with lapatinib and trastuzumab is done, forexample, by the following method or minor variations thereof.Trastuzumab is administered i.v. at a 4 mg/kg loading dose on week 1over 90 minutes, followed by a 2 mg/kg weekly maintenance dosethereafter. Lapatinib is given by mouth either at 1000 mg daily doses orat the one of the dose regimens described in Example 13. MM-111 isadministered as described in the above Examples. For example, MM-111 isadministered i.v. over 90 minutes for the first dose and then weeklyover 60 minutes thereafter.

Example 19: Dosage and Administration of MM-111 with Paclitaxel andTrastuzumab

Administration of MM-111 with paclitaxel and trastuzumab is done, forexample, by the following method or minor variations thereof. Patientsare administered therapy on a 28-day treatment cycle. Paclitaxel dosingbegins on day 1 of cycle 1. Paclitaxel is administered at 80 mg/m²weekly, as an i.v. infusion over 60 minutes. Trastuzumab is administeredat a 4 mg/kg loading dose on week 1, i.v. over 90 minutes, followed by a2 mg/kg weekly maintenance dose thereafter. MM-111 is administered asdescribed in the above Examples. For example, MM-111 is administeredi.v. over 90 minutes for the first dose and then weekly over 60 minutesthereafter.

Example 20: Coadministration of MM-111 and Other Therapeutic Agents

MM-111 (at dosages described herein; see, e.g., Example 15) can beadministered in combination with one or more additional agents to apatient in need thereof for the treatment of a cancer. In particular,MM-111 can be administered in combination with MM-151 (oligoclonalanti-EGFR mixture), TDM-1 (ado-trastuzumab emtansine, an antibody-drugconjugate of the antibody trastuzumab linked to maytansine derivative(DM1)), an mTOR inhibitor (e.g., AZD8055, sirolimus, everolimus,temsirolimus, ridaforolimus, or the dual PI3K/mTOR inhibitor BEZ235),and combinations thereof.

MM-151 is an oligoclonal therapeutic that is a mixture of three fullyhuman monoclonal antibodies designed to bind to non-overlapping epitopesof the epidermal growth factor receptor, or EGFR (also known as ErbB1).An oligoclonal therapeutic is a mixture of two or more distinctmonoclonal antibodies. MM-151 is disclosed, e.g., in copending PCTApplication No. PCT/US12/45235.

MM-111 can be administered in the same dosage form as MM-151, TDM-1,and/or the mTOR inhibitor(s), or the agents can be administered inseparate dosage forms.

In an embodiment, MM-111 and one or more of MM-151, TDM-1, and/or themTOR inhibitor(s) is administered to a patient for the treatment of amalignant tumor, e.g., an ErbB2-expressing or ErbB2 over-expressingtumor (e.g., HER⁺⁺ or HER⁺⁺⁺ tumors). The tumor may be a melanoma, clearcell sarcoma, head and neck, endometrial, prostate, breast, ovarian,gastric, colon, colorectal, lung, bladder, pancreatic, salivary gland,liver, skin, brain or renal tumor.

In another embodiment, MM-111 and MM-151 are co-administered to treat asolid tumor (e.g., an advanced refractory solid tumor) in a patient inneed thereof.

The effect of MM-111 combined with trastuzumab and the dual PI3K/mTORinhibitor BEZ235 on cell proliferation was assessed by the methodsdescribed above or with minor variations thereof in unstimulated andheregulin-stimulated NCI-N87 cells in vitro. Cells were incubated for 2hours with a dose range of MM-111, trastuzumab, or their combination, inthe presence of BEZ235 as shown in FIGS. 14A-B. Cells were plated andeither untreated (Control, open circle), treated with BEZ235 alone (opendiamond), or treated with BEZ235 (5 nM FIG. 14A, 20 nM FIG. 14B) andeither trastuzumab (closed circle), MM-111 (closed square), or thecombination of MM-111 and trastuzumab (closed diamond). Treatment withthe triple combination of MM-111, trastuzumab, and BEZ235 resulted in asignificant reduction in cell number compared to control cells in boththe presence and absence of heregulin stimulation.

The effect of MM-111 combined with the mTOR inhibitor everolimus andtrastuzumab was assessed in unstimulated (FIG. 15A) andheregulin-stimulated (FIG. 15B) BT474M3 cells in vitro. Cells wereplated and either untreated (Control, open circle), treated witheverolimus alone (open diamond), or treated with everolimus (1 nM FIG.15A, 5 nM FIG. 15B) and either trastuzumab (closed circle), MM-111(closed square), or the combination of MM-111 and trastuzumab (closeddiamond). Treatment with the triple combination of MM-111, trastuzumab,and everolimus resulted in a significant reduction in cell numbercompared to control cells in both the presence and absence of heregulinstimulation.

Example 21: Combination Treatment with MM-111 and MEK/PI3k/AKT PathwayInhibitors

MM-111, at dosages described herein (see, e.g., Example 15), can beadministered in combination with one or more MAP/ERK kinase(MEK)/phosphatidylinositol 3-kinase (PI3k)/AKT pathway inhibitors to apatient in need thereof for the treatment of a cancer. MM-111 can beadministered in the same dosage form as the MEK/PI3k/AKT pathwayinhibitor(s) or these agents can be administered in separate dosageforms.

In one embodiment, MM-111 and a MEK/PI3k/AKT pathway inhibitor isadministered to a patient for the treatment of a malignant tumor, e.g.,an ErbB2-expressing or ErbB2 over-expressing tumor (e.g., HER⁺⁺ orHER⁺⁺⁺ tumors). The tumor may be a melanoma, clear cell sarcoma, headand neck, endometrial, prostate, breast, ovarian, gastric, colon,colorectal, lung, bladder, pancreatic, salivary gland, liver, skin,brain or renal tumor.

To determine whether the combination of MM-111 with a MEK inhibitorresults in decreased Erk1/2 activity in a tumor model, MM-111 and theMEK inhibitor trametinib, and were tested in a mouse xenograft model asdescribed below:

Cell Culture and Xenograft Study

BT474-M3 cells are transduced with full length HRG1-β1(Origene)-constructs (encoding PAC-PA-turboGFP for selection) bylentiviral infection. A HRG overexpressing polyclonal cell line(BT474-M3-GFP-HRG) is established after puromycin selection and sortingfor high GFP expressing cell population. A control cell line(BT474-M3-GFP) is engineered to express EGFP in the same manner Cellsare maintained in culture in RPMI supplemented with 10% FBS, penicillin,streptomycin and puromycin. MM-111 is produced in-house at MerrimackPharmaceuticals and MK2206, trametinib, and UO126 (UO126-EtOH) arepurchased from Selleckchem. 7-9 week old female NCRNU—mice (Taconic) areimplanted subcutaneously with 0.72 mg 60-day release 17 β-estradiolpellets (Innovative Research of America).

MM-111 Combination Treatment with Trametinib

48 hours after implantation of the β-estradiol pellets, 15×106BT474-M3-GFP-HRG cells were implanted in the second axillary mammary fatpad. When tumors reached an average volume of 450-500 mm3(day 0), micewere randomized into groups to receive control (phosphate bufferedsaline, intraperitoneally), MM-111 (48 mg/kg, intraperitoneally usingphosphate buffered saline as vehicle), trametinib (3 mg/kg, by oralgavage in 0.5% methylcellulose/0.1% Tween®-80) or a combination ofMM-111 and trametinib. A set (3 per group) of mice are euthanized 4hafter the initial treatment. Tumors were excised and snap-frozen inliquid nitrogen for subsequent protein analysis. Another set of animalscontinued on their assigned treatment regimens for another 7 daysreceiving phosphate buffered saline control (intraperitoneally) on days3 and 6, MM-111 (48 mg/kg, intraperitoneally) on days 3 and 6,trametinib (3 mg/kg, by oral gavage) on days 1, 2, 5 and 6. 24 hoursafter the last treatment, tumors were excised and snap-frozen in liquidnitrogen for subsequent protein analysis. Frozen tumor samples werepulverized and protein lysates were prepared in Tissue ExtractionReagent (Invitrogen) supplemented with Protease Inhibitor Cocktail SetIII (Calbiochem, EMD) and Halt™ Phosphatase Inhibitor Cocktail (ThermoScientific). Lysates were diluted in assay buffer (1% Bovine SerumAlbumin in Tris buffered saline, pH 7.4, containing 0.1% Tween-20) to 1mg/ml concentration and analyzed for phopsho- and total Erk1/2 usingLuminex® immunosandwich assay on a FLEXMAP 3D instrument (Luminex).Antibodies from Cell Signaling Technologies were used to detect totalErk1/2 (3374) and pT202/Y204—sites on Erk1/2 (4370BF). 4h after thestart of treatment, addition of MM-111 to trametinib showed a trend todecreasing Erk1/2 activity (decreasing pT202/Y204-Erk1/2, p=0.086,Student's t-test) compared to treatment with trametinib alone (FIG.16A). 24h after the last treatment, addition of MM-111 to trametinibsignificantly decreased Erk1/2 activity (decreasing pT202/Y204-Erk1/2,p<0.001, Student's t-test) compared to treatment with trametinib alone(FIG. 16B).

Example 22: Combination Treatment with MM-111 and ErbB2-TargetedTherapeutic Agents

MM-111, at dosages described herein (see, e.g., Example 15), can beadministered in combination with one or more additional ErbB2-targetedtherapeutic agents, such as trastuzumab, T-DM1, pertuzumab, etc. Inorder to assess the effect of MM-111 in combination with an additionalErbB2-targeted therapeutic, cells with (FIG. 17B) and without (FIG. 17A)heregulin (HRG) were treated with a dose escalation of T-DM1 and thentreated with MM-111 as follows.

Materials

NCI-N87 cells are transduced with full length HRG1-β1(Origene)-constructs (encoding PAC-PA-turboGFP for selection) bylentiviral infection. A HRG overexpressing polyclonal cell line(NCI-N87-GFP-HRG) is established after puromycin selection and sortingfor high GFP expressing cell population. A control cell line(NCI-N87-GFP) is engineered to express EGFP in the same manner Cells aremaintained in culture in RPMI supplemented with 10% fetal bovine serum,penicillin, streptomycin and puromycin (1 μg/ml).

MM-111 is manufactured at Merrimack Pharmaceuticals. Recombinant humanNRG-1-β1 EGF domain is from R&D Systems. T-DM1 (KADCYLA, Genentech) isobtained from pharmacy. CellTiter-Glo® Luminescent Cell Viability Assayis from Promega.

700 NCI-N87 or BT-474-M3 cells were seeded on 384-well flat clear bottompolystyrene microplates in 20 μl of culture media (RPMI supplementedwith 10% fetal bovine serum and penicillin and streptomycin). Cells wereallowed to adhere 16-20 hours at +37° C., 5% CO₂. Cells were stimulatedfor 4 hours by adding 5 μl of culture media containing recombinant humanNRG-1-β1 EGF domain (HRG1) bringing the final HRG1 stimulationconcentration to 0, 0.313, 0.625, 1.25, 2.5, 5 or 10 nM. After 4 hours,T-DM1 was added in 5 μl of culture media with or without MM-111 bringingthe T-DM1 concentration to 0, 0.001524, 0.004572, 0.013717, 0.041152,0.123457, 0.37037, 1.11111, 3.333333 or 10 μg/ml and the MM-111concentration to 0 or 1 μM. All conditions were assayed in biologicalquadruplicates. Cells were incubated for 72h at +37° C., 5% CO₂. Cellviability was determined by CellTiter-Glo® Luminescent Cell ViabilityAssay by adding 20 μl of reconstituted CellTiter-Glo reagent per well,incubating plated for 10 minutes at RT in an orbital shaker and readingthe luminescence signal using Biotek™ Synergy H1 Hybrid Reader.

700 NCI-N87-GFP or NCI-N87-GFP-HRG1 cells were seeded on 384-well flatclear bottom polystyrene microplates in 20 μl of culture media (RPMIsupplemented with 10% fetal bovine serum and penicillin andstreptomycin). Cells were allowed to adhere 16-20 hours at +37° C., 5%CO2. T-DM1 was added in 5 μl of culture media with or without MM-111bringing the T-DM1 concentration to 0, 0.001524, 0.004572, 0.013717,0.041152, 0.123457, 0.37037, 1.11111, 3.333333 or 10 μg/ml and theMM-111 concentration to 0 or 1 μM. All conditions were assayed inbiological quadruplicates. Cells were incubated for 72h at +37° C., 5%CO₂. Cell viability was determined by CellTiter-Glo® Luminescent CellViability Assay by adding 20 ul of reconstituted CellTiter-Glo reagentper well, incubating plated for 10 minutes at RT in an orbital shakerand reading the luminescence signal using Biotek Synergy H1 HybridReader.

As shown in FIGS. 17A-B, in the absence of endogenous heregulin,treatment of cells with T-DM1 reduces cell viability up to approximately54.6% with 0.1 μg/mL T-DM1, at which point the remaining cells becomeresistant. The addition of MM-111 restores sensitivity of the cells toT-DM1. This effect is significantly increased in the presence ofendogenous heregulin where treatment of cells with T-DM1 reduces cellviability up to approximately 47.3% with 0.04111152 μg/mL T-DM1, atwhich point the remaining cells become resistant. The addition of MM-111restores sensitivity of the cells to T-DM1 further reducing cellviability by approximately 30% to approximately 80% (FIG. 17B).

To test the combination therapy with endogenous heregulin (HRG), NCI-N87cells and BT-474-M3 cells were prepared as described above and treatedwith a dose range of both T-DM1 and heregulin in the presence andabsence of MM-111 (3D graphs, FIGS. 18A-D). Cell viability was testedand numbers are given as % of control and summarized in Tables 1-4below. As can be seen in FIGS. 18A-D, while exogenous HRG decreases theactivity of T-DM1, co-treatment of the cells with a combination ofMM-111 and T-DM1 greatly reduced cell viability in the presence ofendogenous heregulin in both NCI-N87 (FIG. 18B) and BT-474-M3 cells(FIG. 18D).

TABLE 1 NCI-N87 cells without MM-111 HRG T-DM1 (nM) 0 0.001524 0.0045720.013717 0.041152 0.123457 0.37037 1.111111 3.333333 10 (μg/ml) 0100.0001 93.36976 75.62197 51.2155 50.04061 42.64956 46.5761 42.851740.80667 43.84619 0.313 105.2683 100.3208 92.59442 79.62461 74.7190764.7733 50.76463 55.8238 57.24998 57.69454 0.625 110.9354 106.4739106.0159 84.03995 61.45342 71.29415 58.90545 65.075 62.77755 67.861391.25 105.9291 102.2426 94.15791 81.15374 64.90047 66.01914 69.2967569.40256 58.90233 68.76759 2.5 95.5206 90.80036 84.09815 83.7675769.34041 74.67369 67.23904 72.16217 66.05516 65.68811 5 99.9019 94.9823389.09868 80.41984 71.01775 73.54437 68.99217 75.72861 64.94796 63.2151610 114.0075 88.14249 101.4929 80.41022 69.82144 64.32714 61.1348962.15488 59.45985 68.79469

TABLE 2 NCI-N87 cells with MM-111 HRG T-DM1 (nM) 0 0.001524 0.0045720.013717 0.041152 0.123457 0.37037 1.111111 3.333333 10 (μg/ml) 099.99994 95.67672 78.87085 64.23961 46.34934 41.8017 32.86071 35.7953135.56626 32.20002 0.313 101.4158 98.3686 87.83576 74.46012 52.580844.82781 41.52759 37.6864 34.50943 36.02112 0.625 105.024 108.521186.78032 66.85997 55.32827 45.12097 40.0349 38.27817 38.6461 36.362821.25 110.2781 107.8277 99.94741 73.38117 51.00783 43.11336 43.03140.2014 37.52794 35.5527 2.5 118.0867 117.2117 99.28302 90.1342263.70836 52.75964 43.73032 41.26843 36.41708 38.52877 5 126.5145128.6171 118.6616 95.04545 70.74315 51.52235 43.87818 43.0434 42.5410440.95163 10 146.1417 140.3695 122.2993 95.17032 70.81566 52.8167447.2081 43.46258 42.60197 42.12973

TABLE 3 BT-474-M3 cells without MM-111 HRG T-DM1 (nM) 0 0.0015240.004572 0.013717 0.041152 0.123457 0.37037 1.111111 3.333333 10 (μg/ml)0 100 113.2862 98.62592 94.0068 59.46435 46.28945 46.03028 40.9416938.39982 37.44111 0.313 119.8347 116.4401 115.5001 120.231 97.7532998.67097 94.19504 95.98138 95.80208 84.02226 0.625 123.4093 117.8323116.7982 112.1619 108.0231 104.9562 107.4638 97.33409 94.15624 94.017211.25 116.9189 108.1483 107.0419 108.9035 104.0392 109.1902 91.6741694.82696 100.5096 88.67155 2.5 114.6797 113.1709 104.357 114.8767108.3647 96.44198 100.0466 99.7191 99.2052 89.80503 5 117.0592 123.3681119.6607 111.0487 111.2081 98.30082 102.3885 99.45587 90.43802 99.3972310 116.0498 130.2768 115.6098 111.159 104.5468 101.7859 99.51807 101.38998.00674 87.40396

TABLE 4 BT-474-M3 cells with MM-111 HRG T-DM1 (nM) 0 0.001524 0.0045720.013717 0.041152 0.123457 0.37037 1.111111 3.333333 10 (μg/ml) 0100.0001 105.1766 103.9555 103.6039 77.39243 66.65368 57.99249 52.8184561.04489 49.52486 0.313 116.4349 118.4495 115.8389 106.7296 91.7899968.56924 61.57601 59.03206 58.89703 52.39394 0.625 123.4726 120.7381123.5142 115.1022 91.45484 68.63193 61.65257 53.11713 54.6567 47.242961.25 132.3855 131.1941 131.9924 125.6464 100.4235 78.41731 64.3872354.49712 56.83025 54.51656 2.5 131.572 136.6084 146.7699 134.055109.0098 80.11692 73.45718 64.84097 55.26672 53.33503 5 146.0121152.0284 151.8639 138.9854 116.4752 86.2649 70.99611 74.74774 61.7362855.30379 10 157.2453 154.9672 149.674 145.9666 121.6431 91.7930781.23282 69.08484 71.97691 61.17057

Endnotes

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth.

All patents patent applications and publications mentioned herein areincorporated by reference to the same extent as if each independentpatent or patent application was specifically and individually indicatedto be incorporated by reference in its entirety. In particular, WO2012/116317 is incorporated herein by reference in its entirety.

APPENDIX Anticancer Agents

The Table and Appendix below describe effective anti-estrogen agents,receptor tyrosine kinase inhibitors; MEK/PI3 kinase/AKT inhibitors, andmTOR inhibitors that can be used in the methods and compositions of theinvention.

The bispecific anti-ErbB2/anti-ErbB3 antibody co-administered incombination with an agent selected from i) an effective amount of ananti-estrogen agent; ii) an effective amount of a receptor tyrosinekinase inhibitor; iii) a MEK/PI3 kinase/AKT inhibitor; iv) MM-151; v) anmTOR inhibitor; and/or vi) trastuzumab or T-DM1, and combinationsthereof, can be further co-administered with at least a thirdantineoplastic agent selected from any of those disclosed in the Tableand Appendix below.

TABLE 5 Exemplary antineoplastic agents for treatment of breast cancerin combination with a bispecific anti-ErbB2/anti-ErbB3 antibody.Therapeutic Class Exemplary Agent (Generic/Tradename) Exemplary DoseMitotic Inhibitors paclitaxel (TAXOL ®; ABRAXANE ®) 175 mg/m² docetaxel(TAXOTERE ®) 60-100 mg/m² Topoisomerase Inhibitors camptothecintopotecan hydrochloride (HYCAMTIN ®) etoposide (EPOSIN ®) AlkylatingAgents cyclophosphamide (CYTOXAN ®) 600 mg/m² Platinum-Based AgentsCisplatin 20-100 mg/m² carboplatin (PARAPLATIN ®) 300 mg/m² nedaplatin(AQUPLA ®) oxaliplatin (ELOXATIN ®) 65-85 mg/m² satraplatin (SPERA ®)triplatin tetranitrate Selective Estrogen Modulators (SERM) tamoxifen(NOLVADEX ®) 20-40 mg/day raloxifene (EVISTA ®) 60 mg/day toremifene(FARESTON ®) Antimetabolites methotrexate 40 mg/m² Fluorouracil (5-FU)500 mg/m² Raltitrexed Antitumor Antibiotics Doxorubicin (ADRIAMYCIN ®)40-75 mg/m² epirubicin (ELLENCE ®) 60-120 mg/m² Aromatase Inhibitorsaminoglutethimide (CYTADREN ®) 250-2000 mg/day anastrozole (ARIMIDEX ®)1 mg/day letrozole (FEMARA ®) 2.5 mg/day Vorozole exemestane(AROMASIN ®) 25-50 mg/day Testolactone fadrozole (AFEMA ®) Anti-VEGFAgents bevacizumab (AVASTIN ®) 10 mg/kg Anti-ErbB2 (HER2/neu) Agentstrastuzumab (HERCEPTIN ®) 2-8 mg/kg Pertuzumab (OMNITARG ®) Anti-ErbB3(HER3) Agents U3-1287 (AMG 888)

APPENDIX ANTICANCER AGENTS Other anticancer agents for combination witha bispecific anti-ErbB2/anti-ErbB3 antibody Brand Name(s)Manufacturer/Proprietor Anti-IGF1R Antibodies AMG 479 (fully humanizedmAb) Amgen IMCA12 (fully humanized mAb) ImClone NSC-742460 Dyax 19D12(fully humanized mAb) CP751-871 (fully humanized mAb) Pfizer H7C10(humanized mAb) alphaIR3 (mouse) scFV/FC (mouse/human chimera) EM/164(mouse) MK-0646, F50035 Pierre Fabre Medicament, Merck Small MoleculesTargeting IGF1R NVP-AEW541 Novartis BMS-536,924 (1H-benzoimidazol-2-yl)-Bristol-Myers Squibb 1H-pyridin-2-one) BMS-554,417 Bristol-Myers SquibbCycloligan TAE226 PQ401 Anti-EGFR Antibodies INCB7839 Incyte BevacizumabAvastin ® Genentech Cetuximab Erbitux ® IMCLONE mAb 806 Matuzumab(EMD72000) Nimotuzumab (TheraCIM) Panitumumab Vectibix ® Amgen MM-151Merrimack Sym004 Symphogen Zalutumumab Humax Anti-ErbB3 Therapeutics — —U3-1287/AMG888 U3 Pharma/Amgen MM-121 Merrimack PharmaceuticalsAnti-ErbB2 Therapeutics — — trastuzumab Herceptin ® Genentech HKI-272 -neratinib Wyeth KOS-953 - tanespimycin Kosan Biosciences T-DM1 -ado-trastuzumab emtansine Kadcyla ® Genentech Her/ErbB DimerizationInhibitors 2C4, R1273 - Pertuzumab Omnitarg ® Genentech, Roche SmallMolecules Targeting EGFR CI-1033 (PD 183805) Pfizer, Inc. EKB-569Gefitinib IRESSA ™ AstraZeneca Lapatinib (GW572016) GlaxoSmithKlineLapatinib Ditosylate Tykerb ® SmithKline Beecham Erlotinib HCl (OSI-774)Tarceva ® OSI Pharms PD158780 PKI-166 Novartis Tyrphostin AG 1478(4-(3-Chloroanillino)- 6,7-dimethoxyquinazoline) Afatinib (BIBW 2992)Boehringer Ingelheim Small Molecules Targeting MEK CI-1040 (PD184352)AZD6244 (selumetinib) RDEA119 (BAY 869766) GSK1120212 (trametinib) GlaxoSmith Kline PD-0325901 GDC-0973 Genentech UO126-EtOH Cell SignalingTechnology Anti-cMet Antibody Therapies AVEO (AV299) AVEO AMG102 Amgen5D5 (OA-5D5) Genentech Small Molecules Targeting cMet PHA665752ARQ-650RP ArQule ARQ 197 ArQule Alkylating Agents BCNU→ 1,3-bist2-chloroethyl)- nitrosourea Bendamustine Busulfan MyleranGlaxoSmithKline Carboplatin Paraplatin Bristol-Myers Squibb CarboquoneCarmustine CCNU→ 1,-(2-chloroethyl)-3-cyclohexyl- 1-nitrosourea (methylCCNU) Chlorambucil Leukeran ® Smithkline Beecham Chlormethine Cisplatin(Cisplatinum, CDDP) Platinol Bristol-Myers Cyclophosphamide CytoxanBristol-Myers Squibb Dacarbazine (DTIC) Neosar Teva ParenteralFotemustine Hexamethylmelamine (Altretamine, HMM) Hexalen ® MGI Pharma,Inc. Ifosfamide Mitoxana ® ASTA Medica Lomustine Mannosulfan MelphalanAlkeran ® GlaxoSmithKline Nedaplatin Nimustine Oxaliplatin Eloxatin ®Sanofi-Aventis US Prednimustine, Matulane Sigma-Tau Pharmaceuticals,Inc. Procarbazine HCL Ribonucleotide Reductase Inhibitor (RNR)Ranimustine Satraplatin Semustine Streptozocin Temozolomide TreosulfanTriaziquone Triethylene Melamine ThioTEPA Bedford, Abraxis, TevaTriplatin tetranitrate Trofosfamide Uramustine Antimetabolites5-azacytidine Flourouracil (5-FU)/Capecitabine 6-mercaptopurine(Mercaptopurine, 6-MP) 6-Thioguanine (6-TG) Purinethol ® Teva CytosineArabinoside (Cytarabine, Thioguanine ® GlaxoSmithKline Ara-C)Azathioprine Azasan ® AAIPHARMA LLC Capecitabine XELODA ® HLR (Roche)Cladribine (2-CdA, 2- Leustatin ® Ortho Biotech chlorodeoxyadenosine)5-Trifluoromethyl-2′-deoxyuridine Fludarabine phosphate Fludara ® BayerHealth Care Floxuridine (5-fluoro-2) FUDR ® Hospira, Inc. Methotrexatesodium Trexall Barr Pemetrexed Alimta ® Lilly Pentostatin Nipent ®Hospira, Inc. Raltitrexed Tomudex ® AstraZeneca Tegafur AromatoseInhibitor Ketoconazole Glucocorticoids Dexamethasone Decadron ®Dexasone, Wyeth, Inc. Diodex, Hexadrol, Maxidex Prednisolone PrednisoneDeltasone, Orasone, Liquid Pred, Sterapred ® Immunotherapeutics Alphainterferon Angiogenesis Inhibitor Avastin ® Genentech IL-12→ Interleukin12 IL-2→ Interleukin 2 (Aldesleukin) Proleukin ® Chiron ReceptorTyrosine Kinase Inhibitors AMG 386 Amgen Axitinib ((AG-013736) Pfizer,Inc Bosutinib (SKI-606) Wyeth Brivanib alalinate (BMS-582664) BMSCediranib (AZD2171) Recentin AstraVeneca Dasatinib (BMS-354825)Sprycel ® Bristol-Myers Squibb Imatinib mesylate Gleevec NovartisLestaurtinib (CEP-701) Cephalon Motesanib diphosphate (AMG-706)Amgen/Takeda Nilotinib hydrochloride monohydrate Tasigna ® NovartisPazopanib HCL (GW786034) Armala GSK Semaxanib (SU5416) Pharmacia,Sorafenib tosylate Nexavar ® Bayer Sunitinib malate Sutent ® Pfizer,Inc. Vandetanib (AZD647) Zactima AstraZeneca Vatalanib; PTK-787Novartis; Bayer Schering Pharma XL184, NSC718781 Exelixis, GSKMicrotubule-Targeting Agents Colchicine Docetaxel Taxotere ®Sanofi-Aventis US Ixabepilone IXEMPRA ™ Bristol-Myers Squibb LarotaxelSanofi-aventis Ortataxel Spectrum Pharmaceuticals Nanoparticlepaclitaxel (ABI-007) Abraxane ® Abraxis BioScience, Inc. PaclitaxelTaxol ® Bristol-Myers Squibb Tesetaxel Genta Vinblastine sulfateVelban ® Lilly Vincristine Oncovin ® Lilly Vindesine sulphate Eldisine ®Lilly Vinflunine Pierre Fabre Vinorelbine tartrate Navelbine ® PierreFabre mTOR Inhibitors Deforolimus (AP23573, MK 8669, ARIADPharmaceuticals, Inc Ridaforolimus) Everolimus (RAD001, RAD001C)Certican ®, Afinitor Novartis Sirolimus (Rapamycin) Rapamune ® WyethPharma Temsirolimus (CCI-779) Torisel ® Wyeth Pharma BEZ235 AZD8055Protein Synthesis Inhibitor L-asparaginase Elspar ® Merck & Co.Somatostatin Analogue Octreotide acetate Sandostatin ® NovartisTopoisomerase Inhibitors Actinomycin D Camptothecin (CPT) BelotecanDaunorubicin citrate Daunoxome ® Gilead Doxorubicin hydrochlorideDoxil ® Alza Vepesid ® Bristol-Myers Squibb Etoposide Etopophos Hospira,Bedford, Teva Parenteral, Etc. Irinotecan HCL (CPT-11) Camptosar ®Pharmacia & Upjohn Mitoxantrone HCL Novantrone EMD Serono RubitecanTeniposide (VM-26) Vumon ® Bristol-Myers Squibb Topotecan HCL Hycamtin ®GlaxoSmithKline Chemotherapeutic Agents Adriamycin, 5-Fluorouracil,Cytoxin, Bleomycin, Mitomycin C, Daunomycin, Carminomycin, Aminopterin,Dactinomycin, Mitomycins, Esperamicins Clofarabine, Mercaptopurine,Pentostatin, Thioguanine, Cytarabine, Decitabine, Floxuridine,Gemcitabine (Gemzar), Enocitabine, Sapacitabine Hormonal TherapiesAbarelix Plenaxis ™ Amgen Abiraterone acetate CB7630 BTG plc AfimoxifeneTamoGel Ascend Therapeutics, Inc. Anastrazole Arimidex ® AstraZenecaAromatase inhibitor Atamestane plus toremifene Intarcia Therapeutics,Inc. Arzoxifene Eli Lilly & Co. Asentar; DN-101 Novartis; Oregon Health& Science Univ. Bicalutamide Casodex ® AstraZeneca Buserelin Suprefact ®Sanofi Aventis Cetrorelix Cetrotide ® EMD Serono Exemestane Aromasin ®Pfizer Exemestane Xtane Natco Pharma, Ltd. Fadrozole (CGS 16949A)Flutamide Eulexin ® Schering Flutamide Prostacur Laboratorios Almirall,S.A. Fulvestrant Faslodex ® AstraZeneca Goserelin acetate Zoladex ®AstraZeneca Letrozole Femara ® Novartis Letrozole (CGS20267) FemaraChugai Pharmaceutical Co., Ltd. Letrozole Estrochek JagsonpalPharmaceuticals, Ltd. Letrozole Letrozole Indchemie Health SpecialitiesLeuprolide acetate Eligard ® Sanofi Aventis Leuprolide acetate LeoprilVHB Life Sciences, Inc. Leuprolide acetate Lupron ®/Lupron Depot TAPPharma Leuprolide acetate Viador Bayer AG Megestrol acetate Megace ®Bristol-Myers Squibb Magestrol acetate Estradiol Valerate JagsonpalPharmaceuticals, Ltd. (Delestrogen) Medroxyprogesterone acetate VeraplexCombiphar MT206 Medisyn Technologies, Inc. Nafarelin Nandrolonedecanoate Zestabolin Mankind Pharma, Ltd. Nilutamide Nilandron ® AventisPharmaceuticals Raloxifene HCL Evista ® Lilly Tamoxifen Taxifen YungShin Pharmaceutical Tamoxifen Tomifen Alkem Laboratories, Ltd. Tamoxifencitrate Nolvadex AstraZeneca Tamoxifen citrate Soltamox EUSA Pharma,Inc. Tamoxifen citrate Tamoxifen citrate Sopharma JSCo. SOPHARMAToremifene citrate Fareston ® GTX, Inc. Triptorelin pamoate Trelstar ®Watson Labs Triptorelin pamoate Trelstar Depot Paladin Labs, Inc.Protein Kinase B (PKB) Inhibitors Akt Inhibitor ASTEX Astex TherapeuticsAkt Inhibitors NERVIANO Nerviano Medical Sciences AKT Kinase InhibitorTELIK Telik, Inc. AKT Inhibitor Triciribine AKT DECIPHERA DecipheraPharmaceuticals, LLC Perifosine (KRX0401, D-21266) KeryxBiopharmaceuticals, Inc., AEterna Zentaris, Inc. Perifosine withDocetaxel Keryx Biopharmaceuticals, Inc., AEterna Zentaris, Inc.Perifosine with Gemcitabine AEterna Zentaris, Inc. Perifosine withPaclitaxel Keryx Biopharmaceuticals, Inc, AEterna Zentaris, Inc. ProteinKinase-B inhibitor DEVELOGEN DeveloGen AG PX316 Oncothyreon, Inc. RX0183Rexahn Pharmaceuticals, Inc. RX0201 Rexahn Pharmaceuticals, Inc. VQD002VioQuest Pharmaceuticals, Inc. XL418 Exelixis, Inc. ZEN027 AEternaZentaris, Inc. Phosphatidylinositol 3-Kinase (PI3K) Inhibitors BEZ235Novartis AG BGT226 Novartis AG CAL101 Calistoga Pharmaceuticals, Inc.CHR4432 Chroma Therapeutics, Ltd. Erk/PI3K Inhibitors ETERNA AEternaZentaris, Inc. GDC0941 Genentech Inc./Piramed Limited/Roche Holdings,Ltd. Enzastaurin HCL (LY317615) Enzastaurin Eli LillyLY294002/Wortmannin Eli Lilly PI3K Inhibitors SEMAFORE SemaforePharmaceuticals PX866 Oncothyreon, Inc. SF1126 Semafore PharmaceuticalsVMD-8000 VM Discovery, Inc. XL147 Exelixis, Inc. XL147 with XL647Exelixis, Inc. XL765 Exelixis, Inc. PI-103 Roche/Piramed BKM120Cyclin-dependent kinase inhibitors CYC200, r-roscovitine SeliciclibCyclacel Pharma NSC-649890, L86-8275, HMR-1275 Alvocidib NCI TLR9, CD289IMOxine Merck KGaA HYB2055 Idera IMO-2055 Isis Pharma 1018 ISS DynavaxTechnologies/UCSF PF-3512676 Pfizer Enzyme Inhibitor Lonafarnib(SCH66336) Sarasar SuperGen, U Arizona Anti-TRAIL AMG-655 AeternaZentaris, Keryx Biopharma Apo2L/TRAIL, AMG951 Genentech, Amgen Apomab(fully humanized mAb Genentech Other Imprime PGG Biothera CHR-2797AminopeptidaseM1 Chroma Therapeutics E7820, NSC 719239 Integrin-alpha2Eisai INCB007839 ADAM 17, TACE Incyte CNF2024, BIIB021 Hsp90 Biogen IdecMP470, HPK-56 Kit/Met/Ret Shering-Plough SNDX-275/MS-275 HDAC SyndaxZarnestra, Tipifarnib, R115777 Ras Janssen Pharma Volociximab; Eos200-4, M200 alpha581 integrin Biogen Idec; Eli Lilly/UCSF/PDL BioPharmaApricoxib (TP2001) COX-2 Inhibitor Daiichi Sankyo; Tragara Pharma

1. A method of treating a subject having a malignant tumor, the methodcomprising administering to the subject an effective amount acombination therapy comprising one or more agents selected from thegroup consisting of i) an anti-estrogen agent, ii) a receptor tyrosinekinase inhibitor, iii) a MEK inhibitor, iv) a PI3 kinase inhibitor, v)an AKT inhibitor, vi) an mTOR inhibitor, vii) trastuzumab, viii)ado-trastuzumab emtansine, ix) capecitabine, x) cisplatin, and xi)nab-paclitaxel; and an effective amount of an agent that is a bispecificanti-ErbB2/anti-ErbB3 antibody.
 2. The method of claim 1, wherein theanti-estrogen agent is an estrogen receptor antagonist or an aromataseinhibitor.
 3. The method of claim 2, wherein the estrogen receptorantagonist is fulvestrant or tamoxifen.
 4. The method of claim 2,wherein the aromatase inhibitor is letrozole, exemestane, anastrozole,aminoglutethimide, testolactone, vorozole, formestane, or fadrozole. 5.The method of claim 4, wherein the aromatase inhibitor is letrozole. 6.The method of claim 1, wherein the administration to the subject of thecombination therapy does not create a drug-drug interaction-mediatedtoxicity in the subject.
 7. The method of claim 1, wherein theadministration to the subject of the combination therapy creates asubstantially additive or superadditive effect as compared to theseparate administration of each of the combination therapy agents alone.8. The method of claim 1, wherein the receptor tyrosine kinase inhibitoris erlotinib, afatinib, dasatinib, gefitinib, imatinib, pazopinib,lapatinib, sunitinib, nilotinib, or sorafenib.
 9. The method of claim 8,wherein the receptor tyrosine kinase inhibitor is lapatinib.
 10. Themethod of claim 1, further comprising co-administration to the patientof an effective amount of either or both of capecitabine and cisplatin.11.-17. (canceled)
 18. An aqueous solution comprising a bispecificanti-ErbB2/anti-ErbB3 antibody and one or more of i) an anti-estrogenagent, ii) a receptor tyrosine kinase inhibitor, iii) a MEK inhibitor,iv) a PI3 kinase inhibitor, v) an AKT inhibitor, vi) an mTOR inhibitor,vii) trastuzumab, viii) ado-trastuzumab emtansine, ix) capecitabine, x)cisplatin, and xi) nab-paclitaxel.
 19. (canceled)
 20. The method ofclaim 1, wherein said MEK inhibitor is selumetinib, trametinib, UO126,or PD0325901, said PI3K inhibitor is BKM-120 or GDC-0941, and said AKTinhibitor is triciribine or MK-2206.
 21. (canceled)
 22. The aqueoussolution of claim 18, wherein said MEK inhibitor is selumetinib,trametinib, UO126, or PD0325901, said PI3K inhibitor is BKM-120 orGDC-0941, and said AKT inhibitor is triciribine or MK-2206.
 23. Themethod of claim 1, comprising co-administering the MEK inhibitor and thebispecific anti-ErbB2/anti-ErbB3 antibody.
 24. The method of claim 1,comprising co-administering the PI3K inhibitor and the bispecificanti-ErbB2/anti-ErbB3 antibody.
 25. The method of claim 1, comprisingco-administering the mTOR inhibitor and the bispecificanti-ErbB2/anti-ErbB3 antibody.
 26. The method of claim 1, comprisingco-administering trastuzumab or ado-trastuzumab emtansine and thebispecific anti-ErbB2/anti-ErbB3 antibody.
 27. The method of claim 1,wherein said mTOR inhibitor is BEZ235, AZD8055, everolimus,temsirolimus, sirolimus, or ridaforolimus.
 28. The use of claim 11,wherein said mTOR inhibitor is BEZ235, AZD8055, everolimus,temsirolimus, sirolimus, or ridaforolimus.
 29. The aqueous solution ofclaim 18, wherein said mTOR inhibitor is BEZ235, AZD8055, everolimus,temsirolimus, sirolimus, or ridaforolimus.